Agent, uses and methods for treatment

ABSTRACT

The present invention relates to monoclonal anti-Sortilin antibodies which have been found useful correcting a deficient level progranulin (PGRN). In particular, these antibodies can be used in the treatment of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 16/521,279, filed Jul. 24, 2019, which is a Continuation applicationof U.S. application Ser. No. 15/743,549, filed Jan. 10, 2018, which is anational stage filing under 35 U.S.C. § 371 of International ApplicationNo. PCT/EP2016/066516, filed Jul. 12, 2016, which claims priority toGreat Britain Application No. 1512215.3, filed Jul. 13, 2015, the entirecontents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to monoclonal anti-Sortilin antibodiesuseful in correcting a deficient level of progranulin (PGRN). Inparticular these antibodies can be used in the treatment offrontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS).Furthermore, it anticipated that the monoclonal antibodies may also beuseful to treat neurodegenerative disorders such as Alzheimer's Disease(AD).

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(file name: 0993_ST25.txt, created on 22 Jun. 2016, and having a size of144 kB), which file is herein incorporated by reference in its entirety.This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 14, 2022, is namedH095270067US02-SUBSEQ-JDH and is 146,619 bytes in size.

BACKGROUND OF THE INVENTION

Sortilin is a receptor that has been reported to mediate pro-apoptoticeffects of pro-neurotrophins and to mediate trafficking and sorting ofneurotrophin receptors (Nykjær et al, 2012, Trends Neurosci. 2012;35(4):261-70; Glerup et al, Handb Exp Pharmacol, 2014; 220:165-89, Carloet al, J Mol Med (Berl). 2014 September; 92(9):905-11). A number ofsortilin ligands have been identified including neurotensin for which ahigh affinity binding site was localized by x-ray crystallography toinside a beta propeller tunnel in the sortilin molecule (Quistgaard etal, Nat Struct Mol Biol. 2009 January; 16(1):96-8; Quistgaard et al,Protein Sci. 2014, September; 23(9):1291-300). More recently, sortilinwas shown to function as a high affinity receptor for the growth factorprogranulin (PGRN, Hu et al. Neuron. 2010 Nov. 18; 68(4):654-67.

PGRN ((proepithelin, granulin-epithelin precursor, PC-cell-derivedgrowth factor, acrogranin)) is a secreted glycosylated protein withanti-inflammatory and neurotrophic-like actions (For a recent review,see Nguyen, Trends Endocrinol Metab. 2013 December; 24(12):597-606).PGRN is proteolytically cleaved to granulins, but much remains to belearned regarding the physiological role of PGRN and granulins and theidentity of their receptors. PGRN has been implicated in severalcellular functions including cell cycle regulation and cell motility(He, Z. & Bateman, A., J. Mol. Med. 57:600-612 (2003); Monami, G., etal., Cancer Res. (5(5:7103-7110 (2006)), wound repair, inflammation(Zhu, J., et al., Cell 777:867-878 (2002)), induction of growth factorssuch as vascular endothelial growth factor (VEGF) (Tangkeangsirrsin, W.& Serrero, G, Carcinogenesis 25.1587-1592 (2004)), and tumorigenesis(He, Z. & Bateman, A., J. Mol. Med. 81:600-612 (2003), Monami, G., etal., Cancer Res (5(5:7103-7110 (2006); Serrero, G., Biochem Biophys.Res. Commun. 505-409-413 (2003), Lu, R & Serrero, G., Proc. Natl AcadSci U.SA 98 142-147 (2001); Liau, L M., et al., Cancer Res. 60:1353-1360(2000)). PGRN has been reported to bind the TNF receptor (Tang W et al.,Science 2011, 332(6028):478-84). but this observation has beenchallenged by others (Chen et al., J Neurosci. 2013, 33(21):9202-9213).

The binding of PGRN to sortilin has been mapped to the neurotensin siteand reported to be mediated solely through the PGRN C-terminal domain(Zheng et al. PLoS One. 2011; 6(6):e21023; Lee et al. Hum Mol Genet.2013) in a manner similar to neurotensin and in accordance, neurotensinhas been shown to block the interaction of sortilin with PGRN and otherligands. Upon binding, sortilin mediates lysosomal clearance of PGRN andthereby regulates extracellular PGRN levels (Hu et al. 2010). Thus,knockdown or overexpression of sortilin have been shown to regulateextracellular PGRN levels in cell culture (Carrasquillo et al. Am J HumGenet. 2010 Dec. 10; 87(6):890-7) and in mice, sortilin deficiency wasreported to increase PGRN levels and to restore plasma and brainPGRN-levels in PGRN+/−mice (Hu et al. 2010). Interestingly, a singlenucleotide polymorphism (SNP) near sortilin was associated withdecreased plasma PGRN and increased sortilin mRNA levels (Carrasquilloet al. Am J Hum Genet. 2010 Dec. 10; 87(6):890-7). These observationssuggest that sortilin is a key regulator of extracellular PGRN.

PGRN has been linked to frontotemporal dementia (FTD), a progressivedementia characterized by behavioral and semantic changes, as well asfrontotemporal lobar degeneration (FTLD) and neuronal inclusionscontaining TAR DNA Binding Protein-43 (TDP-43) or tau inclusions (Bakeret al, 2006, Nature. 2006 Aug. 24; 442(7105):916-9; Cruts et al, Nature442: 920-924 (2006); Am J Hum Genet. 2010 Dec. 10; 87(6):890-7, M et al,Trends in Genetics 24: 186-194 (2008)). The majority of sporadic andfamilial FTD cases show TDP-43 pathology (˜50%) similar to ALS andFTD-TDP43 and ALS are by some considered to constitute a diseasespectrum (Ito D Neurology. 2011 Oct. 25; 77(17):1636-43; Boxer Ala. etal, Alzheimers Dement. 2013 March; 9(2):176-88; Rademakers et al, NatRev Neurol. 2012 August; 8(8): 423-434) due to common pathologies andgenetic factors and some overlap in symptomatology. No disease-modifyingtreatment options are available for FTD. A subset of frontotemporaldementia patients with TDP-43 pathology have loss of function mutationsin the granulin gene (GRN) resulting in PGRN haplo-insufficiency. Todate, 69 different mutations in the granulin gene, all resulting inreduced PGRN levels and/or function, have been associated with FTD andit is believed that raising extracellular PGRN in plasma and brain wouldcounteract the disease process.

PGRN mutations have also been linked with Alzheimer's disease (AD)(Sheng et al., 2014, Gene. 2014 Jun. 1; 542(2):141-5; Brouwers et al.,2008, Neurology. 2008 Aug. 26; 71(9):656-64) suggesting that PGRNdeficiency may play an important role in AD pathogenesis. Furthermore,neuroprotective effects of PGRN in mouse AD models have been observed(Minami et al, 2014, Nat Med. 2014 October; 20(10):1157-64) providingsupport for the view that enhanced PGRN may be beneficial in AD andpossibly other neurodegenerative conditions.

The present application describes the generation and identification ofanti-human Sortilin antibodies which can regulate PGRN in cellularmodels and in mice. Those antibodies surprisingly bind to a region onSortilin which is distant to the previously reported progranulin bindingsite, the so-called neurotensin-site, and yet are capable of inhibitingSortilin-PGRN interaction and of thereby increasing extracellular PGRN.

The inventors have defined six Sortilin binding regions and surprisinglyidentified that the most efficacious antibodies bind a region (“regionD”). As PGRN has neuroprotective and anti-inflammatory effects, theinventors' findings indicate that such antibodies targeting Sortilin arelikely to have a beneficial effect in the treatment of a range ofneurodegenerative disorders including FTD/FTLD. A subgroup of thesepatients carry a mutation in the gene encoding PGRN leading tohaploinsufficiency. Sortilin antibodies are therefore likely to havesimilar therapeutic benefits for patients suffering from other TDP-43proteinopathies and diseases in which PGRN levels may influenceTDP43-function and pathology, including ALS and AD.

SUMMARY OF INVENTION

The inventors of the present invention have generated monoclonalantibodies which are able to inhibit the binding of PGRN to Sortilin,and which surprisingly bind to a novel Sortilin region denominated the“D-region” as defined in SEQ ID NO:170. Several antibodies identified bythe inventors have properties similar to the D-region antibodies andexperimental evidence indicates that they also bind within the D-region,and those antibodies are herein referred to as D+ antibodies.Accordingly, in one aspect, the invention relates to such antibodies, tocompositions and/or kits comprising such antibodies, and to methods anduses thereof.

The invention also relates to a method of preventing or treating adisease associated with decreased PGRN levels in the brain of a patient,comprising administering an effective dosage of an antibody or anantigen-binding fragment thereof that binds to the D-region of Sortilin.These diseases include i.a. FTD, ALS and TDP43 proteinopathies such asAD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an overview of the generation of region assignment ofhuman antibodies based on Sortilin region binding, effect onPGRN-binding and PGRN-levels and on cross-blocking between theantibodies.

Sortilin-binding antibodies were selected and assigned to regions A-Ebased on binding to shuffle constructs in which the sequencecorresponded to the tetraodon Sortilin sequence within selected regionsof the protein (Example 1, FIGS. 2A-2C).

20 antibodies which inhibited Sortilin-PGRN binding (as measured by HTRFanalysis) were selected (see Example 10, FIG. 5 and FIG. 6 ). 15 ofthese antibodies were D-region antibodies while 3 were D+ antibodies(FIG. 6 ). Subsequent cross-blocking analysis showed that the 18D-region and D+ antibodies (D+ antibodies have a different bindingpattern to shuffle constructs than D-region antibodies. Nevertheless D+antibodies cannot with certainty be assigned to a binding region A-E asoutlined above but shared functional characteristics (cell assays etc.)similar to the D-region antibodies), all cross-blocked each othersupporting that they interacted with the same Sortilin region (Example9, FIGS. 7A-7C). When sortilin antibodies of other region classes weretested in the HTRF sortilin-PGRN binding assay, only two of 41antibodies exhibited an inhibitory effect. One of these two antibodiescross-blocked with the D and D+, but had an atypical shuffle constructbinding pattern (D-like except that it bound the hB01-05 region), whilethe other antibody did not cross-block with the other antibodies thatinhibited PGRN-sortilin binding, thus supporting the conclusion that itbinds to another sortilin region.

These observations show that antibodies binding to an area in sortilindefined by the D-region have the potential to inhibit sortilin-PGRNbinding.

19 cross-blocking antibodies, of which 18 were D-region and D+antibodies, increased extracellular PGRN in a cellular assay (Example13, FIG. 10 and FIG. 11 ). Three of these antibodies were tested in vivoand found to increase plasma PGRN (FIGS. 13A-13C, Example 15).

The boxes in FIG. 1 illustrate steps in the selection of antibodies. A-Erefer to the regions to which the respective Sortilin-binding antibodieswere assigned based on shuffle constructs as described in Example 1 andSEQ ID NOs:171-179. “Other” refers to an antibody which could not beassigned to one region, and which may bind at the interface between theA- and B-regions. Tet refers to antibodies binding alsotetraodon-Sortilin.

In addition to the human antibodies shown, a set of mouse anti-humansortilin antibodies was generated and similarly characterized. Two ofthese antibodies were assigned to the D Region and shown to cross blockwith human D-region and D+ antibodies, to inhibit sortilin-PGRN bindingand to increase extracellular PGRN (see FIG. 4 ).

FIGS. 2A-2C show the region assignment of antibodies based on binding toSortilin shuffle constructs.

FIG. 2A shows a linear illustration of the shuffle constructs used forregion assignment of antibodies as described in Example 1. Sortilinshuffle constructs were generated based on the human Sortilin sequence(SEQ ID NO:169) (sections depicted in grey) in which amino acid residueswere exchanged to the corresponding amino acid from the tetraodonSortilin sequence (depicted in black) (SEQ ID NO:173) (Examples 1-3).

FIG. 2B shows predicted structure of the shuffle constructs illustratedlinearly in A. Dark residues indicate residues changed to thecorresponding tetraodon sequence in the shuffle constructs.

FIG. 2C illustrates the binding pattern of antibodies assigned to theD-region and the E region classes respectively. A “+” indicates bindingto a given shuffle construct and a “−” indicates lack of binding. Basedon the binding pattern to the different shuffle constructs, antibodieswere assigned to regions. The resultant antibody region classes areindicated by A-E. For the illustrated D and E region antibodies, bothbound the human sequences (all grey) as indicated by “+” and neitherbound the tetraodon sequence (all black) as indicated by “−”, whereasthe E region antibody bound the hB45678 shuffle construct while the DRegion antibody did not bind resulting in the localisation of binding asillustrated in FIG. 2A. For D Region antibodies, binding to thefollowing shuffle regions was observed: hsort, hB06-10, B12390. Theantibodies did not bind to hB01-05, B45678, tet. For D+ antibodies,binding to the following shuffle region was observed: hsort, B12390. Theantibodies did not bind to hB01-05, hB06-10, B45678, tet. The F bindingpattern was similar to the D binding pattern except that no binding tothe hB06-10 was observed for D+ antibodies.

The antibodies did not bind to the fully tetraodon Sortilin protein,except two. The two antibodies capable of binding the tetraodon sequencewere denoted “tet”. “Other” refers to an antibody which could not beassigned to one region.

FIG. 3 shows the binding affinities of human D-region and D+ antibodies.Binding affinities to sortilin shuffle constructs by bioLayerinterferometry using Octet 384RED as described in Example 8 (EC50,ng/ml). No shading indicates EC50 of 0.1-10 ng/ml, light grey shadingindicates EC50 >10 ng/ml and grey shading indicates no binding (NB).Region assignment was based on binding patterns is illustrated in FIGS.2A-2C. Shuffle constructs are illustrated in FIGS. 2A-2C and sequencesare given in SEQ ID NOs:171-179. mAb=monoclonal antibody.

FIG. 4 shows the binding affinities of mouse anti-human antibodies toSortilin shuffle constructs as obtained by bioLayer interferometry usingOctet 384RED as described in Example 8 (EC50, ng/ml). No shadingindicates binding and grey shading indicates no binding (NB). Regionassignment based on binding patterns is illustrated in FIGS. 2A-2C.

FIG. 5 shows the effect of Sortilin antibody on Sortilin PGRN binding.The D Region Sortilin human monoclonal (humAb) antibody 45 (filledcircles) prevented PGRN binding to Sortilin, in contrast to a controlSortilin E region antibody (filled triangles) and an IgG control,IgG1-b12 (open triangles) that did not interfere with the binding. Thebinding of antibodies was determined by measuring the displacement ofPGRN binding to Sortilin using Homogenous Time Resolved Fluorescent(HTRF) (Example 10). Dose-response evaluation of antibodies wasperformed with ten concentrations covering 50 μM to 1 μM in a 3-folddilution curve. The half-maximal inhibitory concentration (IC50) valueswere calculated by non-linear regression using sigmoidal concentrationresponse (variable slope) in XLfit 4 (IDBS, UK).

FIG. 6 Summary of effect of antibodies on Sortilin-PGRN bindingdetermined by homogenous time resolved fluorescent (HTRF) analysis asshown in FIG. 5 . In total, 62 antibodies were tested—15 D-regionantibodies and 3 D+ antibodies were found to inhibit sortilin-PGRNbinding and the IC50 values were determined. For two additionalantibodies (E and other regions), an inhibitory effect was observed. Allremaining antibodies were negative in the test. * antibody too weak tofit a dose-response curve. 6% inhibition at 1 μM. ** control (ctrl)antibody too weak to fir a dose-response curve. 37% inhibition at 1 PM.

These observations show that sortilin antibodies characterized by theirD-region or D+ assignment inhibit sortilin's binding to PGRN directlyand are capable of inhibiting sortilin-PGRN binding.

FIGS. 7A-7C show cross-blocking between antibodies. Human antibodies andthe mouse antibodies were all tested in a single experiment where eachantibody was bound to human wild type (WT) Sortilin (FIGS. 7A-7C).Subsequently all other antibodies were tested for binding to thepreformed sortilin:antibody complex (Example 9). The selected 15D-region and 3 D+ human antibodies (based on their effect in the HTRFPGRN-sortilin assay, (FIG. 5 and FIG. 6 ) and two mouse D Regionantibodies all inhibited binding of each other to human WT Sortilin.

The antibodies did not cross-block with antibodies designated to otherregion classes (as illustrated for A-region, E-region and tetraodonrecognizing antibodies numbered AbA1-x, AbE1-x and Abtet in the tablerespectively) except for one cross blocking A region antibody, oneantibody with unknown region assignment (“other”) and a partial blockfor a D+ antibody 548. These data support that the D-region and D+antibodies capable of inhibiting sortilin-PGRN binding in the HTRF assayall interact with the same region in sortilin.

Cross blocking between Sortilin antibodies from the same or differentregions (regions based on binding to shuffle constructs as illustratedin FIGS. 2A-2C) was determined by analyzing interference withantibody-Sortilin binding. Binding of antibodies to Sortilin-ECD-His wasmeasured by BioLayer Interferometry using Octet 384RED (Example 9). Theleft column indicates primary (immobilized) antibodies and the top rowindicates the secondary antibodies (antibodies being tested against theimmobilized antibodies). Binding of both the primary and secondaryantibodies to Sortilin-ECD-His would results in a response value higherthan 0.1 and indicate that both antibodies were binding to differentregions of the protein. Response value less than 0.1 shows lack ofbinding of the secondary antibody and an effective cross blocking by theimmobilized (primary) antibody, which suggests that both antibodies bindto the same region of Sortilin.

FIG. 8 shows the effect of D-region and D+ Sortilin antibodies on thebinding of the selective small molecule ligand AF38469 to Sortilin. Thebinding site for AF38469 has been shown to be similar to the bindingsite of neurotensin and characterized by X-ray crystallography (Schrçderet al. Bioorg Med Chem Lett. 2014 Jan. 1; 24(1):177-80). PGRN has beenreported to bind to the same site (Lee et al. Hum Mol Genet. 2013)antibodies 45 and 68, binding to D-region, and D+ respectively, did notinhibit the binding of AF38469 to sortilin. This data suggests thatthese antibodies have a binding site for Sortilin distinct from thebinding site for AF38469. Therefore, antibodies 45 and 68 inhibitPGRN-sortilin binding through a binding site distinct from the hithertopresumed PGRN binding site in sortilin.

FIG. 9 Effect of antibodies 45 and 68 on cellular binding andendocytosis of PGRN (Example 12). Antibodies 45 and 68 inhibited thebinding and/or endocytosis of PGRN by sortilin overexpressing cells.Addition of neurotensin (NT, 10 uM) similarly reduced binding orendocytosis of PGRN as reflected in reduced fluorescence as expectedwhereas the isotype control antibody B12 did not influence PGRNfluorescence levels.

Antibodies (100 nM) to be tested were added to S18 cells 30 min beforeaddition of recombinant PGRN for 4 hr. The cells were then fixed,stained for PGRN and analyzed by Cellomics. PGRN fluorescence wasmeasured as mean fluorescence per cell. Data is presented as mean±SD.Data analyzed by one-way Anova followed by Dunnett's analysis, allgroups were compared to PGRN. *p<0.05; **p<0.01

FIG. 10 Extracellular PGRN levels estimated by ELISA in media fromcultures of sortilin over-expressing HEK cells (S18). Sortilin D-region(45, 811) and D+ (68) antibodies increased PGRN levels and a similareffect of the sortilin ligand neurotensin was observed whereas thecontrol antibody B12 had no effect. These observations indicate thatD-region and D+ Sortilin antibodies were capable of inhibitingsortilin-mediated clearance of PGRN thereby increasing extracellularPGRN. All antibodies were tested at 100 nM. Neurotensin was tested at 10uM. PGRN levels have been normalized to control. Data is presented asmean±SD. Data was analyzed by one-way Anova followed by Dunnett'sanalysis, all groups were compared to CTRL *p<0.05; **p<0.01. (Example13).

FIG. 11 shows the effect of antibodies on extracellular PGRN in humanSortilin over-expressing HEK cells measured by ELISA as described inexample 13. All selected D-region antibodies and the three selected D+antibodies increased extracellular PGRN. PGRN levels were analysed thesame as above. PGRN levels are normalized to untreated controls andgiven in %. Two antibodies were raised in mouse against human Sortilin(1F2F4 & 5E1F6) and the rest are human antibodies. Ab=monoclonalantibody.

FIG. 12 shows the effect of Sortilin antibody on extracellular PGRN inneuronally differentiated iPSC cells (Example 14). The sortilin D-regionantibody 45 and the D+ antibody 68 increased PGRN levels whereas thecontrol antibodies B12 and anti-HEL had no effect.

Neuronally differentiated iPSC cells were plated into 96 wells plate.One week later, antibodies were added to the cells. Media from the cellswere collected at 48 hrs or 96 hrs and analysed by human PGRN ELISA(Enzo Life sciences) and samples analysed as per the manufacturer'sinstructions. Sortilin human antibodies 45 and 68 increased PGRN levelsin the media at both time points. Control isotype antibodies B12 andAnti-Hel (negative control) did not change extracellular PGRN. Data ispresented as mean±SD. Data was analyzed by one-way Anova followed byDunnett's analysis *p<0.05; **p<0.01 (Example 14)

FIGS. 13A-13C show plasma PGRN levels in human Sortilin expressing knockin (KI) mice treated with Sortilin human antibody (Example 15). Sortilinantibody 45 increased plasma PGRN levels whereas the control antibodyhad no effect.

FIG. 13A shows results of a Time course study: Increased plasma levelsof PGRN were observed after injection of antibody 45 (D Region). Micewere injected with 45 (n=5) or control (n=3) antibody sc at a dose of 10mg/kg. Each group was sacrificed at different time points. In micetreated with control antibodies (Anti-Hel) there was no change in plasmaPGRN, whereas in mice treated with 45 there was a gradual increase inPGRN levels. Effect appeared to peak between 24 and 48 hrs and graduallydecreased by day 4-7.

FIG. 13B shows results of a Subchronic study: Mice were treated twice aweek with 10 mg/kg of 45 and control antibody (Anti-Hel). Samples werecollected from cheek blood every week. Plasma PGRN was elevated at week1 and remained at approximately the same level throughout the entirestudy as compared to the animals treated with control antibody (n=20).

FIG. 13C shows results of a Dose response study: Different doses (4doses: 0.1, 0.4, 2 and 10 mg/kg) of the Sortilin (45) and controlantibodies (Anti-Hel) were injected and mice were sacrificed on day 2.Plasma PGRN was elevated in mice treated with 45 (10 and 2 mg/kg). lowerdoses (0.4 and 0.1 mg/kg) did not have an effect on plasma PGRN. Data ispresented as mean±SD. Data was analyzed by two-way Anova followed byBonferroni's analysis *p<0.05; **p<0.01; ***p<0.001 (Example 15).

FIG. 14 provides an illustration of sortilin regions, based on sortilinshuffle constructs, imposed onto the predicted sortilin structure. Theregions constitute the sections of the sortilin protein in which thechange of selected amino acid residues from the human sequence to thetetraodon sequence inhibited binding for antibodies of that regionclass. The arrow indicates the reported high affinity binding site ofneurotensin and PGRN (Quistgaard Nat Struct Mol Biol. 2009 January;16(1):96-8, Lee et al, Hum Mol Genet. 2013).

FIG. 15A-15I show representative peptides covering the conformationalepitope of antibody 45, 68 and 811. All of the shown peptides show aprotection from exchange larger than 0.5D, except peptide 115-125.Peptide 115-125 is an example of a peptide which is unaffected by thepresence of antibody 45, 68 or 811, and thereby is not a part of theconformational binding epitope (Example 16)

FIGS. 16A-16I show representative peptides covering the conformationalepitope of antibody 30. All of the shown peptides show a protection fromexchange larger than 0.5D, except peptide 563-572, peptide 646-656 andpeptide 704-714. These three peptides are examples of peptides which areunaffected by the presence of antibody 30, and thereby is not a part ofthe conformational binding (Example 16)

FIG. 17 shows an illustration of the microdialysis for procedure.

FIG. 18A shows a time course: effect of systemic administration ofantibody 45 or PBS (50 mg/kg, 10 ml/kg, s.c.) 24 h prior to themicrodialysis experiments on the levels of PRGN in the hippocampus offreely moving hSORT1 mice over time (24 h). (Example 17)

FIG. 18B shows the results of a pooled 24 h dialysis: basal PRGN in thehippocampus of freely moving hSORT1 mice in mab #45- and PBS-treatedmice, 3.3±0.3 ng/ml and 1.1±0.1 ng/ml, respectively, as assessed bypush-pull microdialysis (Example 17).

FIG. 18C presents a table showing PRGN levels (means±SEM) in thehippocampus of freely moving hSORT1 mice measured every 2 h during 24 h,1d after the animals were treated with mab #45 (n=10) or PBS (n=8)(Example 17).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “Sortilin” is synonymous with the Sortilinprotein (identified in for example UniProt as Q99523, 1 and 2). Theamino acid numbering of Sortilin is given with respect to SEQ ID NO:169as shown below, Met being amino acid 1:

MERPWGAADG LSRWPHGLGL LLLLQLLPPS TLSQDRLDAPPPPAAPLPRW SGPIGVSWGL RAAAAGGAFP RGGRWRRSAPGEDEECGRVR DFVAKLANNT HQHVFDDLRG SVSLSWVGDSTGVILVLTTF HVPLVIMTFG QSKLYRSEDY GKNFKDITDLINNTFIRTEF GMAIGPENSG KVVLTAEVSG GSRGGRIFRSSDFAKNFVQT DLPFHPLTQM MYSPQNSDYL LALSTENGLWVSKNFGGKWE EIHKAVCLAK WGSDNTIFFT TYANGSCKADLGALELWRTS DLGKSFKTIG VKIYSFGLGG RFLFASVMADKDTTRRIHVS TDQGDTWSMA QLPSVGQEQF YSILAANDDMVFMHVDEPGD TGFGTIFTSD DRGIVYSKSL DRHLYTTTGGETDFTNVTSL RGVYITSVLS EDNSIQTMIT FDQGGRWTHLRKPENSECDA TAKNKNECSL HIHASYSISQ KLNVPMAPLSEPNAVGIVIA HGSVGDAISV MVPDVYISDD GGYSWTKMLEGPHYYTILDS GGIIVAIEHS SRPINVIKFS TDEGQCWQTYTFTRDPIYFT GLASEPGARS MNISIWGFTE SFLTSQWVSYTIDFKDILER NCEEKDYTIW LAHSTDPEDY EDGCILGYKEQFLRLRKSSV CQNGRDYVVT KQPSICLCSL EDFLCDFGYYRPENDSKCVE QPELKGHDLE FCLYGREEHL TTNGYRKIPGDKCQGGVNPV REVKDLKKKC TSNFLSPEKQ NSKSNSVPIILAIVGLMLVT VVAGVLIVKK YVCGGRFLVH RYSVLQQHAEANGVDGVDAL DTASHTNKSG YHDDSDEDLLE

As used herein, the term “D Region” is intended to refer to the regionon Sortilin (corresponding to residues 523-610 of SEQ ID NO:169)consisting of the amino acids in SEQ ID NO:170 as shown below:

HYYTILDSGG IIVAIEHSSR PINVIKFSTD EGQCWQTYTFTRDPIYFTGL ASEPGARSMN ISIWGFTESF LTSQWVSYTI DFKDILER

For D Region antibodies, binding to the following shuffle regions wasobserved: hsort, hB06-10, B12390. The antibodies did not bind tohB01-05, B45678, tet. For D+ antibodies, binding to the followingshuffle regions was observed: hsort, B12390. The antibodies did not bindto hB01-05, hB06-10, B45678, tet. For the antibodies named “D+” asimilar binding pattern was observed as for “D” region antibodies exceptthat no binding to the hB06-10 was observed for D+ antibodies. Despitethe different binding pattern to shuffle constructs, D+ antibodiesshared functional characteristics (cell assays etc.) to D-regionantibodies.

Certain D and D+ binding antibodies binds particular the region asdefined in SEQ ID NO:185, 186 or 187. Thus, the invention relates incertain embodiments to antibodies or antigen binding fragments thereofthat binds to the D region sequences of SEQ ID 170, and within thatregion SEQ ID NO:185, 186 or 187. By binding, these antibodies orantigen-binding fragments, influence the PGRN levels and thus may beused to treat disease associated with PGRN, such as FTD, ALS and AD.

By further analysing the binding of the D and D+ antibodies of theinvention, partial binding to the neighbouring region (the A region) wasidentified for some of the antibodies. This region corresponds to aminoacids 78-254 of SEQ ID NO:169, as shown in SEQ ID NO:180 and below:

SAPGEDEECG RVRDFVAKLA NNTHQHVFDD LRGSVSLSWVGDSTGVILVL TTFHVPLVIM TFGQSKLYRS EDYGKNFKDITDLINNTFIR TEFGMAIGPE NSGKVVLTAE VSGGSRGGRIFRSSDFAKNF VQTDLPFHPL TQMMYSPQNS DYLLALSTEN GLWVSKNFGG KWEEIHKThis region has been given the term “A region”.

Thus, in certain embodiments the invention relates to antibodies orantigen binding fragments thereof that binds the D region as definedabove and in SEQ ID Nos 170, 185, 186 or 187, and further have affinityfor the A region identified in SEQ ID NO 180. Within the A regioncertain antibodies or antigen binding fragments thereof have affinityfor the A region amino acids identified in SEQ ID NOs 181, 182, 183 or184.

PGRN (proepithelin, granulin-epithelin precursor, PC-cell-derived growthfactor, acrogranin) encodes a 68.5 kDa secreted glycoprotein that has7.5 repeats of smaller granulin motifs, ranging from 6-25 kDa, which canbe proteolytically cleaved from the precursor PGRN (He, Z. & Bateman,A., J. Mol. Med. 81:600-6X2 (2003)). In non-neuronal cells, PGRN hasbeen associated with a variety of events, such as cell cycle regulationand cell motility (He, Z. & Bateman, A., J. Mol. Med. 57:600-612 (2003);Monami, G., et ah, Cancer Res. (5(5:7103-7110 (2006)), wound repair,inflammation (Zhu, J., et ah, Cell 777:867-878 (2002)), induction ofgrowth factors such as vascular endothelial growth factor (VEGF)(Tangkeangsirrsin, W. & Serrero, G, Carcinogenesis 25.1587-1592 (2004)),and tumorigenesis (He, Z. & Bateman, A., J. Mol. Med. 81:600-612 (2003),Monami, G., et al., Cancer Res (5(5:7103-7110 (2006); Serrero, G.,Biochem Biophys. Res. Commun. 505-409-413 (2003), Lu, R & Serrero, G.,Proc. Natl Acad Sa U.SA 98 142-147 (2001); Liau, L M., et al., CancerRes. 60:1353-1360 (2000)).

PGRN mutations result in haploinsufficiency (Baker, M., et ah, Nature442:916-919 (2006); Cruts, M., et ah, Nature 442:920-924 (2006)) and areknown to be present in nearly 50% of familial FTD cases, making PGRNmutation a major genetic contributor to FTD (Cruts, M. & VanBroeckhoven, C, Trends Genet. 24:186-194 (2008); Le Ber, I., et ah,Brain 129:3051-3065 (2006)). The loss-of-function heterozygous characterof PGRN mutations implies that in healthy individuals, PGRN expressionplays a dose-dependent, critical role in protecting healthy individualsfrom the development of FTD.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule or according to some embodiments of theinvention, a fragment of an immunoglobulin molecule which has theability to bind to an epitope of a molecule (“antigen”). Naturallyoccurring antibodies typically comprise a tetramer which is usuallycomposed of at least two heavy (H) chains and at least two light (L)chains. Each heavy chain is comprised of a heavy chain variable domain(abbreviated herein as VH) and a heavy chain constant domain, usuallycomprised of three domains (CH1, CH2 and CH3). Heavy chains can be ofany isotype, including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes), IgA(IgA1 and IgA2 subtypes), IgM and IgE. Each light chain is comprised ofa light chain variable domain (abbreviated herein as VL) and a lightchain constant domain (CL). Light chains include kappa chains and lambdachains. The heavy and light chain variable domain is typicallyresponsible for antigen recognition, while the heavy and light chainconstant domain may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (C1q) of the classicalcomplement system. The VH and VL domains can be further subdivided intoregions of hypervariability, termed “complementarity determiningregions,” that are interspersed with domains of more conserved sequence,termed “framework regions” (FR). Each VH and VL is composed of three CDRDomains and four FR Domains arranged from amino-terminus tocarboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.The variable domains of the heavy and light chains contain a bindingdomain that interacts with an antigen. Of particular relevance areantibodies and their antigen-binding fragments that have been “isolated”so as to exist in a physical milieu distinct from that in which it mayoccur in nature or that have been modified so as to differ from anaturally occurring antibody in amino acid sequence.

The term “epitope” means an antigenic determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and linear epitopes aredistinguished in that the binding to the former, but not the latter, isalways lost in the presence of denaturing solvents. The epitope maycomprise amino acid residues directly involved in the binding and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen-binding peptide (in other words, the amino acidresidue is within the footprint of the specifically antigen-bindingpeptide).

As used herein, the term “antigen-binding fragment of an antibody” meansa fragment, portion, region or domain of an antibody (regardless of howit is produced (e.g., via cleavage, recombinantly, synthetically, etc.))that is capable of binding to an epitope, and thus the term“antigen-binding” is intended to mean the same as “epitope-binding” sothat, for example, an “antigen-binding fragment of an antibody” isintended to be the same as an “epitope-binding fragment of an antibody”.An antigen-binding fragment may contain 1, 2, 3, 4, 5 or all 6 of theCDR Domains of such antibody and, although capable of binding to suchepitope, may exhibit a specificity, affinity or selectivity toward suchepitope that differs from that of such antibody. Preferably, however, anantigen-binding fragment will contain all 6 of the CDR Domains of suchantibody. An antigen-binding fragment of an antibody may be part of, orcomprise, a single polypeptide chain (e.g., an scFv), or may be part of,or comprise, two or more polypeptide chains, each having anamino-terminus and a carboxyl terminus (e.g., a diabody, a Fab fragment,a Fab₂ fragment, etc.). Fragments of antibodies that exhibitantigen-binding ability can be obtained, for example, by proteasecleavage of intact antibodies. More preferably, although the two domainsof the Fv fragment, VL and VH, are naturally encoded by separate genes,or polynucleotides that encode such gene sequences (e.g., their encodingcDNA) can be joined, using recombinant methods, by a flexible linkerthat enables them to be made as a single protein chain in which the VLand VH regions associate to form monovalent antigen-binding molecules(known as single-chain Fv (scFv); see e.g., Bird et al. , (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. (U.S.A.)85:5879-5883). Alternatively, by employing a flexible linker that is tooshort (e.g., less than about 9 residues) to enable the VL and VH domainsof a single polypeptide chain to associate together, one can form abispecific antibody, diabody, or similar molecule (in which two suchpolypeptide chains associate together to form a bivalent antigen-bindingmolecule) (see for instance PNAS USA 90(14), 6444-8 (1993) for adescription of diabodies). Examples of antigen-binding fragmentsencompassed within the present invention include (i) a Fab′ or Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains, or a monovalent antibody as described in WO2007059782; (ii)F(ab′)2 fragments, bivalent fragments comprising two Fab fragmentslinked by a disulfide bridge at the hinge domain; (iii) an Fd fragmentconsisting essentially of the VH and CH1 domains; (iv) a Fv fragmentconsisting essentially of a VL and VH domains, (v) a dAb fragment (Wardet al., Nature 341, 544-546 (1989)), which consists essentially of a VHdomain and also called domain antibodies (Holt et al; Trends Biotechnol.2003 November; 2i(II):484-90); (vi) camelid or nanobodies (Revets et al;Expert Opin Biol Ther. 2005 January; 5_(I): I 11-24) and (vii) anisolated complementarity determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they may be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH domains pair to form monovalent molecules (known as singlechain antibodies or single chain Fv (scFv), see for instance Bird etal., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85,5879-5883 (1988)). These and other useful antibody fragments in thecontext of the present invention are discussed further herein. It alsoshould be understood that the term antibody, unless specified otherwise,also includes antibody-like polypeptides, such as chimeric antibodiesand humanized antibodies, and antibody fragments retaining the abilityto bind to the antigen (antigen-binding fragments) provided by any knowntechnique, such as enzymatic cleavage, peptide synthesis, andrecombinant techniques. An antibody as generated can possess anyisotype. As used herein, “isotype” refers to the immunoglobulin class(for instance IgG1, IgG2, IgG3 or IgG4) that is encoded by heavy chainconstant domain genes. Such antibody fragments are obtained usingconventional techniques known to those of skill in the art; suitablefragments capable of binding to a desired epitope may be readilyscreened for utility in the same manner as an intact antibody.

The term “bispecific antibody” refers to an antibody containing twoindependent antigen-binding fragments that each target independenttargets. These targets can be epitopes present on different proteins ordifferent epitopes present on the same target. Bispecific antibodymolecules can be made using compensatory amino acid changes in theconstant domains of the HCs of the parent monospecific bivalent antibodymolecules. The resulting heterodimeric antibody contains one Fabscontributed from two different parent monospecific antibodies. Aminoacid changes in the Fc domain leads to increased stability of theheterodimeric antibody with bispecificity that is stable over time.(Ridgway et al., Protein Engineering 9, 617-621 (1996), Gunasekaran etal., JBC 285, 19637-1(2010), Moore et al., MAbs 3:6 546-557 (2011),Strop et al., JMB 420, 204-219 (2012), Metz et al., Protein Engineering25:10 571-580 (2012), Labrijn et al., PNAS 110:113, 5145-5150 (2013),Spreter Von Kreudenstein et al., MAbs 5:5 646-654 (2013)). Bispecificantibodies can also include molecules that are generated using ScFvfusions. Two monospecific scfv are then independently joined to Fcdomains able to form stable heterodimers to generate a single bispecificmolecule (Mabry et al., PEDS 23:3 115-127 (2010). Bispecific moleculeshave dual binding capabilities.

An “anti-Sortilin antibody” or “Sortilin antibody” (used interchangeablyherein, depending on the context wherein its written) is an antibody anantigen-binding fragment thereof which binds specifically to Sortilin,and especially to the Sortilin D Region, SEQ ID NO:170. An anti-Sortilinantibody that binds to the Sortilin D Region will usually bind to aconformational epitope or a linear epitope of 3, 4, 5, 6 or 7consecutive amino acids within the D-Region (for example SEQ ID NOs:185,186 or 187) with an affinity (IC50) at or below 22 nM, such as between22 nM and 1 nM, between 10 nM and 1 nM or between 5 nM and 1 nM.According to some embodiments the anti-Sortilin antibodies may also bindto the A region (SEQ ID NOs:180, 181, 182, 183 or 184) although it'semphasized that their main biological function is believed to beachieved by binding to the D Region.

The binding site identified is rather unique as shown with for examplethe binding of the selective small molecule ligand AF38469 to Sortilin.The binding site for AF38469 has been shown to be similar to the bindingsite of neurotensin and characterized by X-ray crystallography (Schroderet al. Bioorg Med Chem Lett. 2014 Jan. 1; 24(1):177-80). PGRN has beenreported to bind to the same site (Lee et al. Hum Mol Genet. 2013).Antibodies 45 and 68, binding to D-region, and D+ respectively, did notinhibit the binding of AF38469 to sortilin. This data suggests thatthese antibodies have a binding site for Sortilin distinct from thebinding site for AF38469 and neurotensin. Therefore, in certainembodiments the invention relates to an antibody, or an antigen-bindingfragment thereof, capable of specifically binding to Sortilin andinhibiting the binding of PGRN to Sortilin, but which binding does notinhibit or substantially inhibit the binding of neurotensin or AF38469to Sortilin. This can be shown using for example displacement of bindingto Sortilin using a scintillation proximity assay (SPA) (Example 11).One way of explaining this finding could be that the antibodies, orantigen-binding fragments thereof, are binding to surface areas ofSortilin whereas the small molecules like neurotensine are bindinginside the binding pocket.

The term “human antibody” (which may be abbreviated to “humAb” or“HuMab”), as used herein, is intended to include antibodies havingvariable and constant domains derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orduring gene rearrangement or by somatic mutation in vivo).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A conventional monoclonal antibody compositiondisplays a single binding specificity and affinity for a particularepitope. In certain embodiments a monoclonal antibody can be composed ofmore than one Fab domain thereby increasing the specificity to more thanone target. The terms “monoclonal antibody” or “monoclonal antibodycomposition” are not intended to be limited by any particular method ofproduction (e.g., recombinant, transgenic, hybridoma, etc.).

The antibodies of the present invention, and their sortilinantigen-binding fragments will preferably be human or, for example forthe mouse antibodies (denoted 1F2, 5E1), “humanized,” particularly ifemployed for therapeutic purposes. The term “humanized” refer to amolecule, generally prepared using recombinant techniques, having anantigen-binding site derived from an immunoglobulin from a non-humanspecies and a remaining immunoglobulin structure based upon thestructure and/or sequence of a human immunoglobulin. The antigen-bindingsite may comprise either complete non-human antibody variable domainsfused to human constant domains, or only the complementarity determiningregions (CDRs) of such variable domains grafted to appropriate humanframework regions of human variable domains. The framework residues ofsuch humanized molecules may be wild type (e.g., fully human) or theymay be modified to contain one or more amino acid substitutions notfound in the human antibody whose sequence has served as the basis forhumanization. Humanization lessens or eliminates the likelihood that aconstant domain of the molecule will act as an immunogen in humanindividuals, but the possibility of an immune response to the foreignvariable domain remains (LoBuglio, A. F. et al. (1989) “Mouse/HumanChimeric Monoclonal Antibody In Man: Kinetics And Immune Response,”Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Another approach focusesnot only on providing human-derived constant domains, but modifying thevariable domains as well so as to reshape them as closely as possible tohuman form. It is known that the variable domains of both heavy andlight chains contain three complementarity-determining regions (CDRs)which vary in response to the antigens in question and determine bindingcapability, flanked by four framework regions (FRs) which are relativelyconserved in a given species and which putatively provide a scaffoldingfor the CDRs. When nonhuman antibodies are prepared with respect to aparticular antigen, the variable domains can be “reshaped” or“humanized” by grafting CDRs derived from nonhuman antibody on the FRspresent in the human antibody to be modified. Application of thisapproach to various antibodies has been reported by Sato, K. et al.(1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) “ReshapingHuman Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al.(1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,”Science 239:1534-1536; Kettleborough, C. A. et al. (1991) “HumanizationOf A Mouse Monoclonal Antibody By CDR-Grafting: The Importance OfFramework Residues On Loop Conformation,” Protein Engineering4:773-3783; Maeda, H. et al. (1991) “Construction Of Reshaped HumanAntibodies With HIV-Neutralizing Activity,” Human Antibodies Hybridoma2:124-134; Gorman, S. D. et al. (1991) “Reshaping A Therapeutic CD4Antibody,” Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, P. R.et al. (1991) “Reshaping A Human Monoclonal Antibody To Inhibit HumanRespiratory Syncytial Virus Infection in vivo,” Bio/Technology9:266-271; Co, M. S. et al. (1991) “Humanized Antibodies For AntiviralTherapy,” Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. etal. (1992) “Humanization Of An Anti-p185her2 Antibody For Human CancerTherapy,” Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M. S. etal. (1992) “Chimeric And Humanized Antibodies With Specificity For TheCD33 Antigen,” J. Immunol. 148:1149-1154. In some embodiments, humanizedantibodies preserve all CDR sequences (for example, a humanized mouseantibody which contains all six CDRs from the mouse antibodies). Inother embodiments, humanized antibodies have one or more CDRs (one, two,three, four, five, six) which are altered with respect to the originalantibody, which are also termed one or more CDRs “derived from” one ormore CDRs from the original antibody. The ability to humanize an antigenis well known (see, e.g., U.S. Pat. Nos. 5,225,539; 5,530,101;5,585,089; 5,859,205; 6,407,213; 6,881,557).

The term “antibody “XX” is intended to denote an antibody orantigen-binding fragment thereof (for example antibody “5E1”),comprising or consisting of the Light Chain, the Light Chain Variabledomain, or the Light Chain Variable domain CDR1-3, as defined by itsrespective SEQ ID NO, and the Heavy Chain, Heavy Chain Variable Domain,or Heavy Chain Variable Domain CDR1-3 as defined by its respective SEQID NO. In certain embodiments the antibody or antigen-binding fragmentthereof are defined by their entire Heavy Chain Variable Domaincomprising as defined by their SEQ ID NO and their Light Chain VariableDomain as defined by their SEQ ID NO.

The numbering of amino acid residues in this region is according toIMGT®, the international ImMunoGeneTics information System® or, Kabat,E. A., Wu, T. T., Perry, H. M., Gottesmann, K. S. & Foeller, C. (1991).Sequences of Proteins of Immunological Interest, 5th edit., NIHPublication no. 91-3242 U.S. Department of Health and Human Services;Chothia, C. & Lesk, A. M. (1987). Canonical structures For TheHypervariable domains Of Immunoglobulins. J. Mol. Biol. 196, 901-917.

As used herein, an antibody or an antigen-binding fragment thereof issaid to “specifically” bind a region of another molecule (i.e., anepitope) if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity or avidity with thatepitope relative to alternative epitopes. In one embodiment, theantibody, or antigen-binding fragment thereof, of the invention binds atleast 10-fold more strongly to its target (Sortilin) than to anothermolecule; preferably at least 50-fold more strongly and more preferablyat least 100-fold more strongly. Preferably, the antibody, orantigen-binding fragment thereof, binds under physiological conditions,for example, in vivo. Thus, by “specifically binding to Sortilin”, weinclude the ability of the antibody, or antigen-binding fragmentthereof, to bind to Sortilin with such specificity and/or under suchconditions. Methods suitable for determining such binding will be knownto those skilled in the art, and exemplary methods are described in theaccompanying Examples. As used herein, the term “binding” in the contextof the binding of an antibody to a predetermined antigen typicallyrefers to binding with an affinity corresponding to a KD of about 10⁻⁷ Mor less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less whendetermined by for instance surface plasmon resonance (SPR) technology ineither a BIAcore® 3000 or T200instrument using the antigen as the ligandand the antibody as the analyte, and binds to the predetermined antigenwith an affinity corresponding to a KD that is at least ten-fold lower,such as at least 100 fold lower, for instance at least 1,000 fold lower,such as at least 10,000 fold lower, for instance at least 100,000 foldlower than its affinity for binding to a non-specific antigen (e.g.,BSA, casein) other than the predetermined antigen or a closely-relatedantigen. The amount with which the affinity is lower is dependent on theKD of the antibody, so that when the KD of the antibody is very low(that is, the antibody is highly specific), then the amount with whichthe affinity for the antigen is lower than the affinity for anon-specific antigen may be at least 10,000 fold. In particular, theinvention pertains to anti-Sortilin antibodies that exhibit a bindingaffinity corresponding to at or below 22 nM, such as between 22 nM and 1nM, between 10 nM and 1 nM or between 5 nM and 1 nM, when determined by,for instance, bioLayer interferometry using an Octet 384RED (Example 8).

In certain embodiments of the invention the invention relates to anantibody or antigen-binding fragment thereof able to compete with humAbantibody 45 or humAb antibody 68 for binding to Sortilin. In anotherembodiment the invention relates to an antibody or antigen-bindingfragment thereof that is able to compete with antibody 45 for binding tothe D Region of Sortilin as defined in SEQ ID NO:170. Such competitivebinding inhibition can be determined using assays and methods well knownin the art, for example using BIAcore® chips with immobilised humanSortilin and incubating with a reference antibody (such as antibody “45”or “68”) with and without an antibody polypeptide to be tested.Alternatively, a pair-wise mapping approach can be used, in which areference antibody (such as antibody “45” or “68”) is immobilised to thesurface of the BIAcore® chip, human Sortilin antigen is bound to theimmobilised antibody, and then a second antibody is tested forsimultaneous binding ability to human Sortilin (see ‘BIAcore® AssayHandbook’, GE Healthcare Life Sciences, 29-0194-00 AA 05/2012; thedisclosures of which are incorporated herein by reference).

The term “kd” (sec−1 or 1/s), as used herein, refers to the dissociationrate constant of a particular antibody-antigen interaction. Said valueis also referred to as the koff value.

The term “ka” (M−1×sec−1 or 1/Msec), as used herein, refers to theassociation rate constant of a particular antibody-antigen interaction.

The term “KD” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the kd by the ka.

The term “KA” (M−1 or 1/M), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the ka by the kd.

In one embodiment, the invention relates to an antibody, orantigen-binding fragment thereof, which exhibits one or more of thefollowing properties:

-   -   (i) a binding affinity (K_(D)) for Sortilin of between 0.5-10        nM, such as 1-5 nM or 1-2 nM;    -   (ii) capability to reduce and/or inhibit PGRN binding to        Sortilin; (iii) capability to reduce and/or inhibit clearance of        PGRN by Sortilin-expressing cells;    -   (iv) capability to reduce and/or inhibit the endocytosis of PGRN        by Sortilin-expressing cells;    -   (v) capability to increase the amount and/or concentration of        PGRN in the plasma in human-Sortilin-expressing knock-in mice.

The term “capability to reduce and/or inhibit PGRN binding to Sortilin”includes an antibody that has the ability to inhibit binding to PGRN atan IC50 less than 50 nM but preferably between 10 nM and 0.2 nM using atime resolved fluorescence assay (HTFR) disclosed in Example 10.

The term “capability to reduce and/or inhibit clearance of PGRN bySortilin-expressing cells” includes the ability to increase theconcentration of PGRN in the medium by at least 25%, such as between 25%and 500%, between 25% and 400% or between 25% and 200% as measured by anELISA assay as disclosed in Example 13.

The “capability to reduce and/or inhibit the endocytosis of PGRN bySortilin-expressing cells” includes the ability reduce the intracellularconcentration of PGRN by at least 10% but preferably between 20 and 100%as measured by a cellomics based assay as disclosed in Example 12.

The “capability to increase the amount and/or concentration of PGRN inthe plasma in human-Sortilin-expressing knock-in mice” includes theability to increase the concentration of PGRN in the plasma by at least25% but preferably between 50 and 500 percent as measured by an ELISAassay as disclosed in Example 15.

It's envisaged that the capability to increase PGRN in the brain mayalso be assayed by for example microdialysis. Thus by “capability toincrease the amount and/or concentration of PGRN in the brain” includesthe ability to increase the concentration of PGRN in the brain by atleast 25% but preferably between 50 and 500 percent as measured bymicrodialysis.

In some antibodies, only part of a CDR, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting therelevant epitope and not in the SDRs can be identified based on previousstudies (for example residues H60-H65 in CDR H2 are often not required),from regions of Kabat CDRs lying outside Chothia hypervariable loops(see, Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICALINTEREST, National Institutes of Health Publication No. 91-3242;Chothia, C. et al. (1987) “Canonical Structures For The HypervariableRegions Of Immunoglobulins,” J. Mol. Biol. 196:901-917), by molecularmodeling and/or empirically, or as described in Gonzales, N. R. et al.(2004) “SDR Grafting Of A Murine Antibody Using Multiple Human GermlineTemplates To Minimize Its Immunogenicity,” Mol. Immunol. 41:863-872. Insuch humanized antibodies at positions in which one or more donor CDRresidues is absent or in which an entire donor CDR is omitted, the aminoacid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity. However, substitutionscan also cause changes of affinity, and significant reductions inaffinity are preferably avoided. Positions for substitution within CDRsand amino acids to substitute can also be selected empirically.

The fact that a single amino acid alteration of a CDR residue can resultin loss of functional binding (Rudikoff, S. etc. (1982) “Single AminoAcid Substitution Altering Antigen-binding Specificity,” Proc. Natl.Acad. Sci. (USA) 79(6):1979-1983) provides a means for systematicallyidentifying alternative functional CDR sequences. In one preferredmethod for obtaining such variant CDRs, a polynucleotide encoding theCDR is mutagenized (for example via random mutagenesis or by asite-directed method (e.g., polymerase chain-mediated amplification withprimers that encode the mutated locus)) to produce a CDR having asubstituted amino acid residue. By comparing the identity of therelevant residue in the original (functional) CDR sequence to theidentity of the substituted (non-functional) variant CDR sequence, theBLOSUM62.iij substitution score for that substitution can be identified.The BLOSUM system provides a matrix of amino acid substitutions createdby analyzing a database of sequences for trusted alignments (Eddy, S. R.(2004) “Where Did The BLOSUM62 Alignment Score Matrix Come From?,”Nature Biotech. 22(8):1035-1036; Henikoff, J. G. (1992) “Amino acidsubstitution matrices from protein blocks,” Proc. Natl. Acad. Sci. (USA)89:10915-10919; Karlin, S. et al. (1990) “Methods For Assessing TheStatistical Significance Of Molecular Sequence Features By Using GeneralScoring Schemes,” Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul,S. F. (1991) “Amino Acid Substitution Matrices From An InformationTheoretic Perspective,” J. Mol. Biol. 219, 555-565. Currently, the mostadvanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table1 presents the BLOSUM62.iij substitution scores (the higher the scorethe more conservative the substitution and thus the more likely thesubstitution will not affect function). If an antigen-binding fragmentcomprising the resultant CDR fails to bind to Sortilin, for example,then the BLOSUM62.iij substitution score is deemed to be insufficientlyconservative, and a new candidate substitution is selected and producedhaving a higher substitution score. Thus, for example, if the originalresidue was glutamate (E), and the non-functional substitute residue washistidine (H), then the BLOSUM62.iij substitution score will be 0, andmore conservative changes (such as to aspartate, asparagine, glutamine,or lysine) are preferred.

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A +4 −1 −2 −2 0 −1 −1 0−2 −1 −1 −1 −1 −2 −1 +1 0 −3 −2 0 R −1 +5 0 −2 −3 +1 0 −2 0 −3 −2 +2 −1−3 −2 −1 −1 −3 −2 −3 N −2 0 +6 +1 −3 0 0 0 +1 −3 −3 0 −2 −3 −2 +1 0 −4−2 −3 D −2 −2 +1 +6 −3 0 +2 −1 −1 −3 −4 −1 −3 −3 −1 0 −1 −4 −3 −3 C 0 −3−3 −3 +9 −3 −4 −3 −3 −1 −1 −3 −1 −2 −3 −1 −1 −2 −2 −1 Q −1 +1 0 0 −3 +5+2 −2 0 −3 −2 +1 0 −3 −1 0 −1 −2 −1 −2 E −1 0 0 +2 −4 +2 +5 −2 0 −3 −3+1 −2 −3 −1 0 −1 −3 −2 −2 G 0 −2 0 −1 −3 −2 −2 +6 −2 −4 −4 −2 −3 −3 −2 0−2 −2 −3 −3 H −2 0 +1 −1 −3 0 0 −2 +8 −3 −3 −1 −2 −1 −2 −1 −2 −2 +2 −3 I−1 −3 −3 −3 −1 −3 −3 −4 −3 +4 +2 −3 +1 0 −3 −2 −1 −3 −1 +3 L −1 −2 −3 −4−1 −2 −3 −4 −3 +2 +4 −2 +2 0 −3 −2 −1 −2 −1 +1 K −1 +2 0 −1 −3 +1 +1 −2−1 −3 −2 +5 −1 −3 −1 0 −1 −3 −2 −2 M −1 −1 −2 −3 −1 0 −2 −3 −2 +1 +2 −1+5 0 −2 −1 −1 −1 −1 +1 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 +6 −4 −2 −2+1 +3 −1 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 +7 −1 −1 −4 −3 −2 S+1 −1 +1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 +4 +1 −3 −2 −2 T 0 −1 0 −1 −1 −1−1 −2 −2 −1 −1 −1 −1 −2 −1 +1 +5 −2 −2 0 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3−2 −3 −1 +1 −4 −3 −2 +11 +2 −3 Y −2 −2 −2 −3 −2 −1 −2 −3 +2 −1 −1 −2 −1+3 −3 −2 −2 +2 +7 −1 V 0 −3 −3 −3 −1 −2 −2 −3 −3 +3 +1 −2 +1 −1 −2 −2 0−3 −1 +4

The invention thus contemplates the use of random mutagenesis toidentify improved CDRs. In the context of the present invention,conservative substitutions may be defined by substitutions within theclasses of amino acids reflected in one or more of the following threetables:

Amino Acid Residue Classes For Conservative Substitutions:

TABLE 2 Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg(R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn(N), and Gln (Q) Aliphatic Uncharged Residues Cly (G), Ala (A), Val (V),Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), andPro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W)Alternative Conservative Amino Acid Residue Substitution Classes:

TABLE 3 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y WAlternative Physical and Functional Classifications of Amino AcidResidues:

TABLE 4 Alcohol Group-Containing Residues S and T Aliphatic Residues I,L, V and M Cycloalkenyl-Associated Residues F, H, W and Y HydrophobicResidues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively ChargedResidues D and E Polar Residues C, D, E, H, K, N, Q, R, S and TPositively Charged Residues H, K and R Small Residues A, C, D, G, N, P,S, T and V Very Small Residues A, G and S Residues Involved In TurnFormation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible Residues Q,T, K, S, G, P, D, E and R

More conservative substitutions groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2d Ed. 1993), W. H. Freeman and Company.

Phage display technology can alternatively be used to increase (ordecrease) CDR affinity. This technology, referred to as affinitymaturation, employs mutagenesis or “CDR walking” and re-selection usesthe target antigen or an antigenic antigen-binding fragment thereof toidentify antibodies having CDRs that bind with higher (or lower)affinity to the antigen when compared with the initial or parentalantibody (See, e.g. Glaser et al. (1992) J. Immunology 149:3903).Mutagenizing entire codons rather than single nucleotides results in asemi-randomized repertoire of amino acid mutations. Libraries can beconstructed consisting of a pool of variant clones each of which differsby a single amino acid alteration in a single CDR and which containvariants representing each possible amino acid substitution for each CDRresidue. Mutants with increased (or decreased) binding affinity for theantigen can be screened by contacting the immobilized mutants withlabeled antigen. Any screening method known in the art can be used toidentify mutant antibodies with increased or decreased affinity to theantigen (e.g., ELISA) (See Wu et al. 1998, Proc. Natl. Acad. Sci.(U.S.A.) 95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDRwalking which randomizes the Light Chain may be used possible (see,Schier et al., 1996, J. Mol. Bio. 263:551).

Methods for accomplishing such affinity maturation are described forexample in: Krause, J. C. et al. (2011) “An Insertion Mutation ThatDistorts Antibody Binding Site Architecture Enhances Function Of A HumanAntibody,” MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10; Kuan,C. T. et al. (2010) “Affinity-Matured Anti-Glycoprotein NMB RecombinantImmunotoxins Targeting Malignant Gliomas And Melanomas,” Int. J. Cancer10.1002/ijc.25645; Hackel, B. J. et al. (2010) “Stability And CDRComposition Biases Enrich Binder Functionality Landscapes,” J. Mol.Biol. 401(1):84-96; Montgomery, D. L. et al. (2009) “Affinity MaturationAnd Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41,”MAbs 1(5):462-474; Gustchina, E. et al. (2009) “Affinity Maturation ByTargeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab DerivedFrom A Synthetic Naïve Human Antibody Library And Directed Against TheInternal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With ImprovedHIV-1 Neutralization Potency And Breadth,” Virology 393(1):112-119;Finlay, W. J. et al. (2009) “Affinity Maturation Of A Humanized RatAntibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A HighLevel Of Mutational Plasticity Both Inside And Outside TheComplementarity-Determining Regions,” J. Mol. Biol. 388(3):541-558;Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity AndSpecificity For Therapeutic Development,” Methods Mol. Biol.525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation OfHuman GM-CSF Antibodies By Targeted CDR-Diversification,” Mol. Immunol.46(1):135-144; and Barderas, R. et al. (2008) “Affinity Maturation OfAntibodies Assisted By In Silico Modeling,” Proc. Natl. Acad. Sci. (USA)105(26):9029-9034.

Thus, the sequence of CDR variants of encompassed antibodies or theirantigen-binding fragments may differ from the sequence of the CDR of theparent antibody through substitutions; for instance substituted 4 aminoacid residue, 3 amino acid residue, 2 amino acid residue or 1 of theamino acid residues. According to an embodiment of the invention it isfurthermore envisaged that the amino acids in the CDR regions may besubstituted with conservative substitutions, as defined in the below 3tables.

The term “transgenic non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or trans-chromosomes (either integrated or non-integratedinto the animal's natural genomic DNA) and which is capable ofexpressing fully human antibodies. For example, a transgenic mouse canhave a human light chain transgene and either a human heavy chaintransgene or human heavy chain trans-chromosome, such that the mouseproduces human anti-Sortilin antibody when immunized with Sortilinantigen and/or cells expressing Sortilin. The human heavy chaintransgene may be integrated into the chromosomal DNA of the mouse, as isthe case for transgenic mice, for instance HuMAb mice, such as HCo7 orHCo12 mice, or the human heavy chain transgene may be maintainedextra-chromosomally, as is the case for trans-chromosomal KM mice asdescribed in WO02/43478. Such transgenic and trans-chromosomal mice(collectively referred to herein as “transgenic mice”) are capable ofproducing multiple isotypes of human monoclonal antibodies to a givenantigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-Jrecombination and isotype switching.

Transgenic, nonhuman animal can also be used for production ofantibodies against a specific antigen by introducing genes encoding suchspecific antibody, for example by operatively linking the genes to agene which is expressed in the milk of the animal.

The term “treatment” or “treating” as used herein means ameliorating,slowing, attenuating or reversing the progress or severity of a diseaseor disorder, or ameliorating, slowing, attenuating or reversing one ormore symptoms or side effects of such disease or disorder. For purposesof this invention, “treatment” or “treating” further means an approachfor obtaining beneficial or desired clinical results, where “beneficialor desired clinical results” include, without limitation, alleviation ofa symptom, diminishment of the extent of a disorder or disease,stabilized (i.e., not worsening) disease or disorder state, delay orslowing of the progression a disease or disorder state, amelioration orpalliation of a disease or disorder state, and remission of a disease ordisorder, whether partial or total detectable or undetectable.

An “effective amount,” when applied to an antibody or antigen-bindingfragment thereof of the invention, refers to an amount sufficient, atdosages and for periods of time necessary, to achieve an intendedbiological effect or a desired therapeutic result including, withoutlimitation, clinical results. The phrase “therapeutically effectiveamount,” when applied to an antibody or antigen-binding fragment thereofof the invention, is intended to denote an amount of the antibody, orantigen-binding fragment thereof, that is sufficient to ameliorate,palliate, stabilize, reverse, slow, attenuate or delay the progressionof a disorder or disease state, or of a symptom of the disorder ordisease. In an embodiment, the method of the present invention providesfor administration of the antibody, or antigen-binding fragment thereof,in combinations with other compounds. In such instances, the “effectiveamount” is the amount of the combination sufficient to cause theintended biological effect.

A therapeutically effective amount of an anti-Sortilin antibody orantigen-binding fragment thereof of the invention may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the anti-Sortilin antibody orantigen-binding fragment thereof to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

The antibodies are preferably a human or humanized antibody.

The numbering of amino acid residues in this region is according toIMGT®, the international ImMunoGeneTics Information System® or, Kabat,E. A., Wu, T. T., Perry, H. M., Gottesmann, K. S. & Foeller, C. (1991).Sequences of Proteins of Immunological Interest, 5th edit., NIHPublication no. 91-3242 U.S. Department of Health and Human Services;Chothia, C. & Lesk, A. M. (1987). Canonical structures For TheHypervariable domains Of Immunoglobulins. J. Mol. Biol. 196, 901-917.

Antibody 5E1:

Accordingly, the invention relates to an antibody, or an antigen-bindingfragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:1;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:2;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:3;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:4;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:5;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:6.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:8 and the light chain variable domainof SEQ ID NO:7.

Antibody 1F2:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:16 and the light chain variabledomain of SEQ ID NO:15.

Antibody 068:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:24 and the light chain variabledomain of SEQ ID NO:23.

Antibody 1320:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:26;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:27;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:30.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:32 and the light chain variabledomain of SEQ ID NO:31.

Antibody 93-05:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:33;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:34;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:35;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:36;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:37;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:38.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:40 and the light chain variabledomain of SEQ ID NO:39.

Antibody 93-01:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:41;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:42;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:43;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:44;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:45;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:46.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:48 and the light chain variabledomain of SEQ ID NO:47.

Antibody 924:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:49;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:50;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:51;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:52;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:53;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:54.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:56 and the light chain variabledomain of SEQ ID NO:55.

Antibody 1276:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:57;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:58;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:59;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:60;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:61;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:62.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:64 and the light chain variabledomain of SEQ ID NO:63.

Antibody 849:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:65;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:66;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:67;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:68;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:69;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:70.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:72 and the light chain variabledomain of SEQ ID NO:71.

Antibody 531-02:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:73;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:74;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:75;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:76;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:77;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:78.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:80 and the light chain variabledomain of SEQ ID NO:79.

Antibody 548-01:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:81;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:82;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:83;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:84;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:85;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:86.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:88 and the light chain variabledomain of SEQ ID NO:87.

Antibody 548-02:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:89;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:90;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:91;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:92;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:93;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:94.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:96 and the light chain variabledomain of SEQ ID NO:95.

Antibody 1289-02:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:97;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:98;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:99;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:100;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:101;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:102.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:104 and the light chain variabledomain of SEQ ID NO:103.

Antibody 811-02:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:105;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:106;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:107;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:108;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:109;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:110.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:112 and the light chain variabledomain of SEQ ID NO:111.

Antibody 566-01:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:113;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:114;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:115;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:116;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:117;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:118.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:120 and the light chain variabledomain of SEQ ID NO:119.

Antibody 562:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:121;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:122;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:123;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:124;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:125;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:126.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:128 and the light chain variabledomain of SEQ ID NO:127.

Antibody 193:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:129;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:130;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:131;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:132;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:133;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:134.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:136 and the light chain variabledomain of SEQ ID NO:135.

Antibody 88:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:137;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:138;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:139;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:140;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:141;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:142.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:144 and the light chain variabledomain of SEQ ID NO:143.

Antibody 045:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:145;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:146;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:147;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:148;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:149;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:150.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:152 and the light chain variabledomain of SEQ ID NO:151.

Antibody 044:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:153;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:154;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:155;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:156;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:157;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:158.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:160 and the light chain variabledomain of SEQ ID NO:159.

Antibody 002:

According to another embodiment the invention relates to an antibody, oran antigen-binding fragment thereof, comprising or consisting of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:161;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:162;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:163;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:164;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:165;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:166.

Preferably, the monoclonal antibody may comprise or consist of the heavychain variable domain of SEQ ID NO:168 and the light chain variabledomain of SEQ ID NO:167.

The antibodies mentioned above may, according to one embodiment, furthercomprise a variant with no more than 4 amino acid differences, or nomore than 3 amino acid differences, or no more than 2 amino aciddifferences, or no more than 1 amino acid difference from said CDR1,CDR2, and/or CDR3 (VH and/or VL) sequences.

Further, the antibodies may be in a composition together with apharmaceutically acceptable carrier. The antibodies of the invention maybe used in therapy. In particular, the antibodies of the invention maybe used in treating FTD or ALS or TDP43 proteinopathies such asAlzheimer's Disease (AD).

The treatment envisioned by the present invention may be chronic and thepatient may be treated at least 2 weeks, such as at least for 1 month,6, months, 1 year or more.

The antibodies of the present invention may, for example, be monoclonalantibodies produced by the hybridoma method first described by Kohler etal., Nature 256, 495 (1975), or may be monoclonal antibodies produced byrecombinant DNA or other methods. Monoclonal antibodies may also beisolated from phage antibody libraries using the techniques describedin, for example, Clackson et al., Nature 352, 624-628 (1991) and Markset al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may beobtained from any suitable source. Thus, for example, monoclonalantibodies may be obtained from hybridomas prepared from murine splenicB lymphocyte cells obtained from mice immunized with an antigen ofinterest, for instance, in the form of cells expressing the antigen onthe surface, or a nucleic acid encoding an antigen of interest.Monoclonal antibodies may also be obtained from hybridomas derived fromantibody-expressing cells of immunized humans or from non-human mammalssuch as rats, rabbits, dogs, sheep, goats, primates, etc.

In one embodiment, the antibody of the invention is a human antibody.Human monoclonal antibodies directed against Sortilin may be generatedusing transgenic or transchromosomal mice carrying parts of the humanimmune system rather than the mouse system. Such transgenic andtrans-chromosomic mice include mice referred to herein as HuMAb mice andKM mice, respectively.

The HuMAb mouse contains a human immunoglobulin gene minilocus thatencodes unrearranged human heavy variable and constant (p and Y) andlight variable and constant (K) chain immunoglobulin sequences, togetherwith targeted mutations that inactivate the endogenous p and K chainloci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, themice exhibit reduced expression of mouse IgM or K and in response toimmunization, the introduced human heavy and light chain transgenes,undergo class switching and somatic mutation to generate high affinityhuman IgG, K monoclonal antibodies (Lonberg, N. et al. (1994), supra;reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113,49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 1365-93 (1995) and Harding, F. and Lonberg, N., Ann. N. Y. Acad. Sci 764536-546 (1995)). The preparation of HuMAb mice is described in detail inTaylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J.et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J.Immunol. 152, 2912-2920 (1994), Taylor, L. et al., InternationalImmunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology14, 845-851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825,5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318,5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227,WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in theirendogenous light chain (kappa) genes (as described in Chen et al., EMBOJ. 12, 811-820 (1993)), a CMD disruption in their endogenous heavy chaingenes (as described in Example 1 of WO 01/14424), and a KCo5 human kappalight chain transgene (as described in Fishwild et al., NatureBiotechnology 14, 845-851 (1996)). Additionally, the HCo7 mice have aHCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429), the HCo12 mice have a HCo12 human heavy chain transgene (asdescribed in Example 2 of WO 01/14424), the HCo17 mice have a HCo17human heavy chain transgene (as described in Example 2 of WO 01/09187)and the HCo20 mice have a HCo20 human heavy chain transgene. Theresulting mice express human immunoglobulin heavy and kappa light chaintransgenes in a background homozygous for disruption of the endogenousmouse heavy and kappa light chain loci.

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478. HCo12-Balb/c, HCo17-Balb/c and HCo20-Balb/c mice can begenerated by crossing HCo12, HCo17 and HCo20 to KCo5[J/K](Balb) asdescribed in WO 09/097006.

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain trans-chromosomecomposed of chromosome 14 antigen-binding fragment hCF (SC20) asdescribed in WO 02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according towell-known techniques. Human monoclonal or polyclonal antibodies of thepresent invention, or antibodies of the present invention originatingfrom other species may also be generated transgenically through thegeneration of another non-human mammal or plant that is transgenic forthe immunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies may beproduced in, and recovered from, the milk of goats, cows, or othermammals. See for instance U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172and 5,741,957.

The antibody of the invention may be of any isotype. The choice ofisotype typically will be guided by the desired effector functions, suchas ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4.Either of the human light chain constant domains, kappa or lambda, maybe used. If desired, the class of an anti-Sortilin antibody of thepresent invention may be switched by known methods. For example, anantibody of the present invention that was originally IgM may be classswitched to an IgG antibody of the present invention. Further, classswitching techniques may be used to convert one IgG subclass to another,for instance from IgG1 to IgG2. Thus, the effector function of theantibodies of the present invention may be changed by isotype switchingto, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody forvarious therapeutic uses. In one embodiment an antibody of the presentinvention is an IgG1 antibody, for instance an IgG1, K. An antibody issaid to be of a particular isotype if its amino acid sequence is mosthomologous to that isotype, relative to other isotypes.

In one embodiment, the antibody of the invention is a full-lengthantibody, preferably an IgG antibody, in particular an IgG1, K antibody.In another embodiment, the antibody of the invention is an antibodyantigen-binding fragment or a single-chain antibody.

Antibodies and antigen-binding fragments thereof may e.g. be obtained byantigen-binding fragmentation using conventional techniques, andantigen-binding fragments screened for utility in the same manner asdescribed herein for whole antibodies. For example, F(ab′)2antigen-binding fragments may be generated by treating antibody withpepsin. The resulting F(ab′)2 antigen-binding fragment may be treated toreduce disulfide bridges to produce Fab′ antigen-binding fragments. Fabantigen-binding fragments may be obtained by treating an IgG antibodywith papain; Fab′ antigen-binding fragments may be obtained with pepsindigestion of IgG antibody. An F(ab′) antigen-binding fragment may alsobe produced by binding Fab′-described below via a thioether bond or adisulfide bond. A Fab′ antigen-binding fragment is an antibodyantigen-binding fragment obtained by cutting a disulfide bond of thehinge domain of the F(ab′)₂. A Fab′-antigen-binding fragment may beobtained by treating an F(ab′)₂ antigen-binding fragment with a reducingagent, such as dithiothreitol. Antibody antigen-binding fragment mayalso be generated by expression of nucleic acids encoding suchantigen-binding fragments in recombinant cells (see for instance Evanset al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimericgene encoding a portion of an F(ab′)2 antigen-binding fragment couldinclude DNA sequences encoding the CH1 domain and hinge domain of the Hchain, followed by a translational stop codon to yield such a truncatedantibody antigen-binding fragment molecule.

In one embodiment, the anti-Sortilin antibody is a monovalent antibody,preferably a monovalent antibody as described in WO2007059782 (which isincorporated herein by reference in its entirety) having a deletion ofthe hinge region. Accordingly, in one embodiment, the antibody is amonovalent antibody, wherein said anti-Sortilin antibody is constructedby a method comprising: i) providing a nucleic acid construct encodingthe light chain of said monovalent antibody, said construct comprising anucleotide sequence encoding the VL region of a selected antigenspecific anti-Sortilin antibody and a nucleotide sequence encoding theconstant CL region of an Ig, wherein said nucleotide sequence encodingthe VL region of a selected antigen specific antibody and saidnucleotide sequence encoding the CL region of an Ig are operably linkedtogether, and wherein, in case of an IgG1 subtype, the nucleotidesequence encoding the CL region has been modified such that the CLregion does not contain any amino acids capable of forming disulfidebonds or covalent bonds with other peptides comprising an identicalamino acid sequence of the CL region in the presence of polyclonal humanIgG or when administered to an animal or human being; ii) providing anucleic acid construct encoding the heavy chain of said monovalentantibody, said construct comprising a nucleotide sequence encoding theVH region of a selected antigen specific antibody and a nucleotidesequence encoding a constant CH region of a human Ig, wherein thenucleotide sequence encoding the CH region has been modified such thatthe region corresponding to the hinge region and, as required by the Igsubtype, other regions of the CH region, such as the CH3 region, doesnot comprise any amino acid residues which participate in the formationof disulphide bonds or covalent or stable non-covalent inter-heavy chainbonds with other peptides comprising an identical amino acid sequence ofthe CH region of the human Ig in the presence of polyclonal human IgG orwhen administered to an animal human being, wherein said nucleotidesequence encoding the VH region of a selected antigen specific antibodyand said nucleotide sequence encoding the CH region of said Ig areoperably linked together; iii) providing a cell expression system forproducing said monovalent antibody; iv) producing said monovalentantibody by co-expressing the nucleic acid constructs of (i) and (ii) incells of the cell expression system of (iii).

Similarly, in one embodiment, the anti-Sortilin antibody is a monovalentantibody, which comprises:

-   (i) a variable domain of an antibody of the invention as described    herein or an antigen-binding part of the said domain, and-   (ii) a CH domain of an immunoglobulin or a domain thereof comprising    the CH2 and CH3 domains, wherein the CH domain or domain thereof has    been modified such that the domain corresponding to the hinge domain    and, if the immunoglobulin is not an IgG4 subtype, other domains of    the CH domain, such as the CH3 domain, do not comprise any amino    acid residues, which are capable of forming disulfide bonds with an    identical CH domain or other covalent or stable non-covalent    inter-heavy chain bonds with an identical CH domain in the presence    of polyclonal human IgG.

In a further embodiment, the heavy chain of the monovalent antibody hasbeen modified such that the entire hinge region has been deleted.

In another further embodiment, the sequence of the monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

The invention also includes “Bispecific Antibodies,” wherein ananti-Sortilin binding region (e.g., a Sortilin-binding region of ananti-Sortilin monoclonal antibody) is part of a bivalent or polyvalentbispecific scaffold that targets more than one epitope, (for example asecond epitope could comprise an epitope of an active transportreceptor, such that the Bispecific Antibody would exhibit improvedtranscytosis across a biological barrier, such as the Blood BrainBarrier). Thus, in another further embodiment, the monovalent Fab of ananti-Sortilin antibody may be joined to an additional Fab or scfv thattargets a different protein to generate a bispecific antibody. Abispecific antibody can have a dual function, for example a therapeuticfunction imparted by an anti-sortilin binding domain and a transportfunction that can bind to a receptor molecule to enhance transfer crossa biological barrier, such as the blood brain barrier.

Antibodies and antigen-binding fragments thereof of the invention alsoinclude single chain antibodies. Single chain antibodies are peptides inwhich the heavy and light chain Fv domains are connected. In oneembodiment, the present invention provides a single-chain Fv (scFv)wherein the heavy and light chains in the Fv of an anti-Sortilinantibody of the present invention are joined with a flexible peptidelinker (typically of about 10, 12, 15 or more amino acid residues) in asingle peptide chain. Methods of producing such antibodies are describedin for instance U.S. Pat. No. 4,946,778, Pluckthun in The Pharmacologyof Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242,423-426 (1988), Huston et al., PNAS USA 85, 5879-5883 (1988) andMcCafferty et al., Nature 348, 552-554 (1990). The single chain antibodymay be monovalent, if only a single VH and VL are used, bivalent, if twoVH and VL are used, or polyvalent, if more than two VH and VL are used.

The antibodies and antigen-binding fragments thereof described hereinmay be modified by inclusion of any suitable number of modified aminoacids and/or associations with such conjugated substituents. Suitabilityin this context is generally determined by the ability to at leastsubstantially retain the Sortilin selectivity and/or Sortilinspecificity associated with the non-derivatized parent anti-Sortilinantibody. The inclusion of one or more modified amino acids may beadvantageous in, for example, increasing polypeptide serum half-life,reducing polypeptide antigenicity, or increasing polypeptide storagestability. Amino acid(s) are modified, for example, co-translationallyor post-translationally during recombinant production (e.g., N-linkedglycosylation at N-X-S/T motifs during expression in mammalian cells) ormodified by synthetic means. Non-limiting examples of a modified aminoacid include a glycosylated amino acid, a sulfated amino acid, aprenylated (e. g., farnesylated, geranyl-geranylated) amino acid, anacetylated amino acid, an acylated amino acid, a PEGylated amino acid, abiotinylated amino acid, a carboxylated amino acid, a phosphorylatedamino acid, and the like. References adequate to guide one of skill inthe modification of amino acids are replete throughout the literature.Example protocols are found in Walker (1998) Protein Protocols OnCD-Rom, Humana Press, Totowa, N.J. The modified amino acid may, forinstance, be selected from a glycosylated amino acid, a PEGylated aminoacid, a farnesylated amino acid, an acetylated amino acid, abiotinylated amino acid, an amino acid conjugated to a lipid moiety, oran amino acid conjugated to an organic derivatizing agent.

The antibodies and antigen-binding fragments thereof of the invention,may also be chemically modified by covalent conjugation to a polymer tofor instance increase their circulating half-life. Exemplary polymers,and methods to attach them to peptides, are illustrated in for instanceU.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. Additionalillustrative polymers include polyoxyethylated polyols and polyethyleneglycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000and about 40,000, such as between about 2,000 and about 20,000, e.g.,about 3,000-12,000 g/mol).

The antibodies and antigen-binding fragments thereof of the presentinvention may further be used in a diagnostic method or as a diagnosticimaging ligand.

In one embodiment, antibodies and antigen-binding fragments thereof ofthe invention comprising one or more radiolabeled amino acids areprovided. A radiolabeled anti-Sortilin antibody may be used for bothdiagnostic and therapeutic purposes (conjugation to radiolabeledmolecules is another possible feature). Non-limiting examples of suchlabels include, but are not limited to bismuth (²¹³Bi), carbon (¹¹C,¹³C, ¹⁴C), chromium (⁵¹Cr), cobalt (⁵⁷Co, ⁶⁰Co), copper (⁶⁴Cu),dysprosium (¹⁶⁵Dy), erbium (¹⁶⁹Er), fluorine (¹⁸F), gadolinium (¹⁵³Gd,¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga), germanium (⁶⁸Ge), gold (¹⁹⁸Au), holmium(¹⁶⁶Ho), hydrogen (³H), indium (¹¹¹In, ¹¹²In, ¹¹³In, ¹¹⁵In), iodine(¹²¹I, ¹²³I ¹²⁵I, ¹³¹I), iridium (¹⁹²Ir), iron (⁵⁹Fe), krypton(^(81m)Kr), lanthanium (¹⁴⁰La), lutelium (¹⁷⁷Lu), manganese (⁵⁴Mn),molybdenum (⁹⁹Mo), nitrogen (¹³N, ¹⁵N), oxygen (¹⁵O), palladium (¹⁰³Pd),phosphorus (³²P), potassium (⁴²K), praseodymium (¹⁴²Pr), promethium(¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), rubidium (⁸¹Rb, ⁸²Rb),ruthenium (⁸²Ru, ⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium(⁷⁵Se), sodium (²⁴Na), strontium (⁸⁵Sr, ⁸⁹Sr, ⁹²Sr), sulfur (³⁵S),technetium (⁹⁹Tc), thallium (²⁰¹TI), tin (¹¹³Sn, ¹¹⁷Sn), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Yb), yttrium (⁹⁰Y) and zinc (⁶⁵Zn). Methodsfor preparing radiolabeled amino acids and related peptide derivativesare known in the art (see for instance Junghans et al., in CancerChemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo,eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581, 4,735,210,5,101,827, 5,102,990 (U.S. RE35,500), U.S. Pat. Nos. 5,648,471 and5,697,902. For example, a radioisotope may be conjugated by a chloramineT method (Lindegren, S. et al. (1998) “Chloramine-T InHigh-Specific-Activity Radioiodination Of Antibodies UsingN-Succinimidyl-3-(Trimethylstannyl)Benzoate As An Intermediate,” Nucl.Med. Biol. 25(7):659-665; Kurth, M. et al. (1993) “Site-SpecificConjugation Of A Radioiodinated Phenethylamine Derivative To AMonoclonal Antibody Results In Increased Radioactivity Localization InTumor,” J. Med. Chem. 36(9):1255-1261; Rea, D. W. et al. (1990)“Site-specifically radioiodinated antibody for targeting tumors,” CancerRes. 50(3 Suppl):857s-861 s).

The invention also provides anti-Sortilin antibodies and antigen-bindingfragments thereof that are detectably labeled using a fluorescent label(such as a rare earth chelate (e.g., a europium chelate)), afluorescein-type label (e.g., fluorescein, fluorescein isothiocyanate,5-carboxyfluorescein, 6-carboxy fluorescein, dichlorotriazinylaminefluorescein), a rhodamine-type label (e.g., ALEXA FLUOR® 568(Invitrogen), TAMRA® or dansyl chloride), VIVOTAG 680 XL FLUOROCHROME™(Perkin Elmer), phycoerythrin; umbelliferone, Lissamine; a cyanine; aphycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen) or an analoguethereof, all of which are suitable for optical detection.Chemiluminescent labels may be employed (e.g., luminol, luciferase,luciferin, and aequorin). Such diagnosis and detection can also beaccomplished by coupling the diagnostic molecule of the presentinvention to detectable substances including, but not limited to,various enzymes, enzymes including, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase, or to prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin.

Chemiluminescent labels may be employed (e.g., luminol, luciferase,luciferin, and aequorin). Such diagnosis and detection can also beaccomplished by coupling the diagnostic molecule of the presentinvention to detectable substances including, but not limited to,various enzymes, enzymes including, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase, or to prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin. Paramagnetic labelscan also be employed, and are preferably detected using PositronEmission Tomography (PET) or Single-Photon Emission Computed Tomography(SPECT). Such paramagnetic labels include, but are not limited tocompounds containing paramagnetic ions of Aluminum (Al), Barium (Ba),Calcium (Ca), Cerium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu),Gandolinium (Gd), Holmium (Ho), Iridium (Ir), Lithium (Li), Magnesium(Mg), Manganese (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os),Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthenium (Ru),Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm),Tin (Sn), Titanium (Ti), Tungsten (W), and Zirconium (Zi), andparticularly, Co⁺², CR⁺² Cr⁺³, Cu⁺², Fe⁺², Fe⁺³, Ga⁺³, Mn⁺³, Ni⁺², Ti⁺³,V⁺³, and V⁺⁴, positron emitting metals using various positron emissiontomographies, and non-radioactive paramagnetic metal ions.

Thus in one embodiment the anti-Sortilin antibody or Sortilin-bindingfragment thereof of the invention may be labelled with a fluorescentlabel, a chemiluminescent label, a paramagnetic label, a radioisotopiclabel or an enzyme label. The labelled antibody of fragment may be usedin detecting or measuring the presence or amount of said Sortilin in thebrain of a subject. This method may comprise the detection ormeasurement of in vivo imaging of anti-Sortilin antibody orSortilin-binding fragment bound to said Sortilin and may comprises exvivo imaging of said anti-Sortilin antibody or Sortilin-binding fragmentbound to such Sortilin.

In a further aspect, the invention relates to an expression vectorencoding one or more polypeptide chains of an antibody of the inventionor an antigen-binding-domain thereof. Such expression vectors may beused for recombinant production of the antibodies and antigen-bindingfragments of the invention.

An expression vector in the context of the present invention may be anysuitable DNA or RNA vector, including chromosomal, non-chromosomal, andsynthetic nucleic acid vectors (a nucleic acid sequence comprising asuitable set of expression control elements). Examples of such vectorsinclude derivatives of SV40, bacterial plasmids, phage DNA, baculovirus,yeast plasmids, vectors derived from combinations of plasmids and phageDNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-Sortilin antibody-encoding nucleic acid is comprised in a naked DNAor RNA vector, including, for example, a linear expression element (asdescribed in, for instance, Sykes and Johnston, Nat Biotech 12, 355-59(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in, for instance, Schakowski et al., Mol Ther3, 793-800 (2001)), or as a precipitated nucleic acid vector construct,such as a CaPO₄-precipitated construct (as described in, for instance,WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigleret al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic CellGenetics 2, 603 (1981)). Such nucleic acid vectors and the usage thereofare well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and5,973,972).

In one embodiment, the vector is suitable for expression ofanti-Sortilin antibodies or antigen-binding fragments thereof in abacterial cell. Examples of such vectors include expression vectors suchas BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J BiolChem 264, 5503-5509 (1989), pET vectors (Novagen, Madison, Wis.) and thelike).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), Grant et al., Methods in Enzymol 153,516-544 (1987), Mattanovich, D. et al. Methods Mol. Biol. 824, 329-358(2012), Celik, E. et al. Biotechnol. Adv. 30(5), 1108-1118 (2012), Li,P. et al. Appl. Biochem. Biotechnol. 142(2), 105-124 (2007), Böer, E. etal. Appl. Microbiol. Biotechnol. 77(3), 513-523 (2007), van der Vaart,J. M. Methods Mol. Biol. 178, 359-366 (2002), and Holliger, P. MethodsMol. Biol. 178, 349-357 (2002)).

In an expression vector of the invention, anti-Sortilinantibody-encoding nucleic acids may comprise or be associated with anysuitable promoter, enhancer, and other expression-facilitating elements.Examples of such elements include strong expression promoters (e. g.,human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, andHIV LTR promoters), effective poly (A) termination sequences, an originof replication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an antibody or antigen-binding fragment thereof of theinvention as defined herein or a bispecific molecule of the invention asdefined herein. Examples of host cells include yeast, bacteria, andmammalian cells, such as CHO or HEK cells.

For example, in one embodiment, the present invention provides a cellcomprising a nucleic acid stably integrated into the cellular genomethat comprises a sequence coding for expression of an anti-Sortilinantibody of the present invention or an antigen-binding fragmentthereof. In another embodiment, the present invention provides a cellcomprising a non-integrated nucleic acid, such as a plasmid, cosmid,phagemid, or linear expression element, which comprises a sequencecoding for expression of an anti-Sortilin antibody or antigen-bindingfragment thereof of the invention.

In a further aspect, the invention relates to a method for producing ananti-Sortilin antibody of the invention, said method comprising thesteps of a) culturing a hybridoma or a host cell of the invention asdescribed herein above, and b) purifying the antibody of the inventionfrom the culture media.

In one embodiment, the invention relates to a preparation that, as suchterm is used herein, comprises an anti-Sortilin antibody as definedherein, and that is substantially free of naturally-arising antibodiesthat are either not capable of binding to sortilin or that do notmaterially alter the anti-Sortilin functionality of the preparation.Thus, such a preparation does not encompass naturally-arising serum, ora purified derivative of such serum, that comprises a mixture of ananti-Sortilin antibody and another antibody that does not alter thefunctionality of the anti-Sortilin antibody of the preparation, whereinsuch functionality is:

-   (i) a binding affinity (K_(D)) for Sortilin;-   (ii) a capability to reduce and/or inhibit PGRN binding to Sortilin;-   (iii) a capability to reduce and/or inhibit clearance of PGRN by    Sortilin-expressing cells;-   (iv) a capability to reduce and/or inhibit the endocytosis of PGRN    by Sortilin-expressing cells;-   (v) a capability to increase the amount and/or concentration of PGRN    in the plasma in human-Sortilin-expressing knock-in mice; a    capability to increase the amount and/or concentration of PGRN in    the brain and/or-   (vi) a capability, when administered chronically, to provide    treatment of frontotemporal dementia (FTD) and/or amyotrophic    lateral sclerosis (ALS).

The invention particularly relates to preparations of such ananti-Sortilin antibody having a structural change in its amino acidsequence (in any of its CDRs, variable domains, framework residuesand/or constant domains) relative to the structure of anaturally-occurring anti-Sortilin antibody, wherein said structuralchange causes the anti-Sortilin antibody monoclonal antibody to exhibita markedly altered functionality (i.e., more than a 20% difference, morethan a 40% difference, more than a 60% difference, more than an 80%difference, more than a 100% difference, more than a 150% difference,more than a 2-fold difference, more than a 4-fold difference, more thana 5-fold difference, or more than a 10-fold difference in functionality)relative to the functionality exhibited by said naturally-occurringanti-Sortilin antibody; wherein such functionality is:

-   -   (i) a binding affinity (K_(D)) for Sortilin;    -   (ii) a capability to reduce and/or inhibit PGRN binding to        Sortilin;    -   (iii) a capability to reduce and/or inhibit clearance of PGRN by        Sortilin-expressing cells;    -   (iv) a capability to reduce and/or inhibit the endocytosis of        PGRN by Sortilin-expressing cells;    -   (vi) a capability to increase the amount and/or concentration of        PGRN in the plasma in human-Sortilin-expressing knock-in mice;    -   (vii) a capability to increase the amount and/or concentration        of PGRN in the brain and/or    -   (vi) a capability, when administered chronically, to provide        treatment of frontotemporal dementia (FTD), amyotrophic lateral        sclerosis (ALS) and/or Alzheimer's Disease (AD).        especially wherein such altered functionality is a result of the        structural change and thus is inseparable from it.

The term “substantially free” of naturally-arising antibodies refers tothe complete absence of such naturally-arising antibodies in suchpreparations, or of the inclusion of a concentration of suchnaturally-arising antibodies in such preparations that does notmaterially affect the Sortilin-binding properties of the preparations.An antibody is said to be “isolated” if it has no naturally-arisingcounterpart or has been separated or purified from components whichnaturally accompany it.

The term “naturally-arising antibodies,” as it relates to suchpreparations, refers to antibodies (including naturally-arisingautoantibodies) elicited within living humans or other animals, as anatural consequence to the functioning of their immune systems.

Thus, the preparations of the present invention do not exclude, andindeed explicitly encompass, such preparations that contain ananti-Sortilin antibody and a deliberately added additional antibodycapable of binding to an epitope that is not possessed by Sortilin. Suchpreparations particularly include embodiments thereof wherein thepreparation exhibits enhanced efficacy in treating frontotemporaldementia (FTD) and/or amyotrophic lateral sclerosis (ALS).

The antibodies of antigen-binding fragments thereof of the presentinvention may be produced in different cell lines, such as a human cellline, a mammal non-human cell line, and insect cell line, for example aCHO cell line, HEK cell line, BHK-21 cell line, murine cell line (suchas a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line,HKB-11 cell line, CAP cell line and HuH-7 human cell line (Dumont et al,2015, Crit Rev Biotechnol. September 18:1-13., the contents which isincluded herein by reference).

In an even further aspect, the invention relates to a pharmaceuticalcomposition comprising:

(i) an anti-Sortilin antibody or antigen-binding fragment thereof, bothas defined herein, or a preparation, as such term is defined herein,that comprises such an anti-Sortilin antibody or antigen-bindingfragment thereof; and

(ii) a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 22ndEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2013.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosencompound of the present invention and the chosen mode of administration.Suitability for carriers and other components of pharmaceuticalcompositions is determined based on the lack of significant negativeimpact on the desired biological properties of the chosen compound orpharmaceutical composition of the present invention (e.g., less than asubstantial impact (10% or less relative inhibition, 5% or less relativeinhibition, etc.)) on epitope binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a non-ionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition. The diluent is selected to not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers andthe like. The compositions may also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes).

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular compositions of the present invention employed, or the amidethereof, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode, including: parenteral, topical, oral or intranasal means forprophylactic and/or therapeutic treatment. In one embodiment, apharmaceutical composition of the present invention is administeredparenterally. The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includeepidermal, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.Additional suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the art. In one embodiment thatpharmaceutical composition is administered by intravenous orsubcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound of thepresent invention.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Such carriers may include gelatin,glyceryl monostearate, glyceryl distearate, biodegradable, biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid alone or with awax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, micro-emulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe an aqueous or non-aqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays antibody absorption, for example,monostearate salts and gelatin. Sterile injectable solutions may beprepared by incorporating the active compound in the required amount inan appropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses describedherein are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. Parenteral compositions may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

The effective dosages and the dosage regimens for the anti-Sortilinantibodies depend on the disease or condition to be treated and may bedetermined by the persons skilled in the art. An exemplary, non-limitingrange for a therapeutically effective amount of an antibody of thepresent invention is about 0.1-10 mg/kg/body weight, such as about 0.1-5mg/kg/body weight, for example about 0.1-2 mg/kg/body weight, such asabout 0.1-1 mg/kg/body weight, for instance about 0.15, about 0.2, about0.5, about 1, about 1.5 or about 2 mg/kg/body weight.

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the anti-Sortilin antibody employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acomposition of the present invention will be that amount of the compoundwhich is the lowest dose effective to produce a therapeutic effect. Suchan effective dose will generally depend upon the factors describedabove. Administration may e.g. be intravenous, intramuscular,intraperitoneal, or subcutaneous, and for instance administered proximalto the site of the target. If desired, the effective daily dose of apharmaceutical composition may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalcomposition as described above.

The labelled antibodies or antigen-binding fragments thereof of theinvention can be used for diagnostic purposes to detect, diagnose, ormonitor diseases or disorders. The invention provides for the detectionor diagnosis of a neurodegenerative or cognitive disease or disorder,including but not limited to FTD, ALS or TDP43 proteinopathies such asAlzheimer's Disease (AD), comprising: (a) assaying the existence ofpyroglutamated Aβ fragments in cells or tissue samples of a subjectusing one or more antibodies that specifically bind to Sortilin; and (b)comparing the level of the antigen with a control level, e.g. levels innormal tissue samples, whereby an increase in the assayed level ofantigen compared to the control level of antigen is indicative of thedisease or disorder, or indicative of the severity of the disease ordisorder.

The antibodies or antigen-binding fragments thereof of the invention canbe used to assay Sortilin or antigen-binding fragments of Sortilin in abiological sample using immuno-histochemical methods well-known in theart. Other antibody-based methods useful for detecting protein includeimmunoassays such as the enzyme linked immunoassay (ELISA) and theradioimmunoassay assay (RIA) and mesoscale discovery platform basedassays (MSD). Suitable antibody labels may be used in such kits andmethods, and labels known in the art include enzyme labels, such asalkaline phosphatase and glucose oxidase; radioisotope labels, such asiodine (¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(¹²¹In), and technetium (^(99m)Tc); and luminescent labels, such asluminol and luciferase; and fluorescent labels, such as fluorescein andrhodamine.

The presence of labeled anti-Sortilin antibodies or theirSortilin-binding fragments may be detected in vivo for diagnosticpurposes. In one embodiment, diagnosis comprises: a) administering to asubject an effective amount of such labeled molecule; b) waiting for atime interval following administration to allow the labeled molecule toconcentrate at sites (if any) of Aβ deposition and to allow for unboundlabeled molecule to be cleared to background level; c) determining abackground level; and d) detecting the labeled molecule in the subject,such that detection of labeled molecule above the background level isindicative that the subject has the disease or disorder, or isindicative of the severity of the disease or disorder. In accordancewith such embodiment, the molecule is labeled with an imaging moietysuitable for detection using a particular imaging system known to thoseskilled in the art. Background levels may be determined by variousmethods known in the art, including comparing the amount of labeledantibody detected to a standard value previously determined for aparticular imaging system. Methods and systems that may be used in thediagnostic methods of the invention include, but are not limited to,computed tomography (CT), whole body scan such as positron emissiontomography (PET), magnetic resonance imaging (MRI), and sonography.

In a further aspect, the invention relates to an antibody, orantigen-binding fragment thereof, of the invention, for use in medicine.

In a further aspect, the invention relates to an antibody, orantigen-binding fragment thereof, of the invention, for use in treatinga disease associated with decreased PGRN levels in the brain of apatient.

In a further aspect, the invention relates to the use of the antibody,or antigen-binding fragment thereof, of the invention, in themanufacture of a medicament for treating a disease associated withdecreased PGRN levels in the brain of a patient.

In a further aspect, the invention relates to a method of preventing ortreating a disease associated with decreased PGRN levels in the brain ofa patient, comprising administering an effective dosage of an antibodyof the invention, or an antigen-binding fragment thereof.

It is preferred that in the uses and methods of those aspects of theinvention the disease is: FTD; ALS; or TDP43 proteinopathies, such asAD.

Preferably, in the uses and methods of those aspects of the invention,the treatment is chronic, and is preferably for at least 2 weeks, suchas at least for 1 month, 6, months, 1 year or more.

In a further aspect, the invention provides a kit comprising theantibody, or antigen-binding fragment thereof, of the invention.

TABLE 5 Antibody Sequences Ab 5E1 Seq ID No: 1 5E1 CDR1 Light Chain SeqID No: 2 5E1 CDR2 Light Chain Seq ID No: 3 5E1 CDR3 Light Chain Seq IDNo: 4 5E1 CDR1 Heavy Chain Seq ID No: 5 5E1 CDR2 Heavy Chain Seq ID No:6 5E1 CDR3 Heavy Chain Seq ID No: 7 5E1 VL Seq ID No: 8 5E1 VH Ab 1F2Seq ID No: 9 1F2 CDR1 Light Chain Seq ID No: 10 1F2 CDR2 Light Chain SeqID No: 11 1F2 CDR3 Light Chain Seq ID No: 12 1F2 CDR1 Heavy Chain Seq IDNo: 13 1F2 CDR2 Heavy Chain Seq ID No: 14 1F2 CDR3 Heavy Chain Seq IDNo: 15 1F2 VL Seq ID No: 16 1F2 VH Ab 068 Seq ID No: 17 068 CDR1 LightChain Seq ID No: 18 068 CDR2 Light Chain Seq ID No: 19 068 CDR3 LightChain Seq ID No: 20 068 CDR1 Heavy Chain Seq ID No: 21 068 CDR2 HeavyChain Seq ID No: 22 068 CDR3 Heavy Chain Seq ID No: 23 068 VL Seq ID No:24 068 VH Ab 1320 Seq ID No: 25 1320 CDR1 Light Chain Seq ID No: 26 1320CDR2 Light Chain Seq ID No: 27 1320 CDR3 Light Chain Seq ID No: 28 1320CDR1 Heavy Chain Seq ID No: 29 1320 CDR2 Heavy Chain Seq ID No: 30 1320CDR3 Heavy Chain Seq ID No: 31 1320 VL Seq ID No: 32 1320 VH Ab 93-05Seq ID No: 33 93-05 CDR1 Light Chain Seq ID No: 34 93-05 CDR2 LightChain Seq ID No: 35 93-05 CDR3 Light Chain Seq ID No: 36 93-05 CDR1Heavy Chain Seq ID No: 37 93-05 CDR2 Heavy Chain Seq ID No: 38 93-05CDR3 Heavy Chain Seq ID No: 39 93-05 VL Seq ID No: 40 93-05 VH Ab 93-01Seq ID No: 41 93-01 CDR1 Light Chain Seq ID No: 42 93-01 CDR2 LightChain Seq ID No: 43 93-01 CDR3 Light Chain Seq ID No: 44 93-01 CDR1Heavy Chain Seq ID No: 45 93-01 CDR2 Heavy Chain Seq ID No: 46 93-01CDR3 Heavy Chain Seq ID No: 47 93-01 VL Seq ID No: 48 93-01 VH Ab 924Seq ID No: 49 924 CDR1 Light Chain Seq ID No: 50 924 CDR2 Light ChainSeq ID No: 51 924 CDR3 Light Chain Seq ID No: 52 924 CDR1 Heavy ChainSeq ID No: 53 924 CDR2 Heavy Chain Seq ID No: 54 924 CDR3 Heavy ChainSeq ID No: 55 924 VL Seq ID No: 56 924 VH Ab 1276 Seq ID No: 57 1276CDR1 Light Chain Seq ID No: 58 1276 CDR2 Light Chain Seq ID No: 59 1276CDR3 Light Chain Seq ID No: 60 1276 CDR1 Heavy Chain Seq ID No: 61 1276CDR2 Heavy Chain Seq ID No: 62 1276 CDR3 Heavy Chain Seq ID No: 63 1276VL Seq ID No: 64 1276 VH Ab 849 Seq ID No: 65 849 CDR1 Light Chain SeqID No: 66 849 CDR2 Light Chain Seq ID No: 67 849 CDR3 Light Chain Seq IDNo: 68 849 CDR1 Heavy Chain Seq ID No: 69 849 CDR2 Heavy Chain Seq IDNo: 70 849 CDR3 Heavy Chain Seq ID No: 71 849 VL Seq ID No: 72 849 VH Ab531-02 Seq ID No: 73 531-02 CDR1 Light Chain Seq ID No: 74 531-02 CDR2Light Chain Seq ID No: 75 531-02 CDR3 Light Chain Seq ID No: 76 531-02CDR1 Heavy Chain Seq ID No: 77 531-02 CDR2 Heavy Chain Seq ID No: 78531-02 CDR3 Heavy Chain Seq ID No: 79 531-02 VL Seq ID No: 80 531-02 VHAb 548-01 Seq ID No: 81 548-01 CDR1 Light Chain Seq ID No: 82 548-01CDR2 Light Chain Seq ID No: 83 548-01 CDR3 Light Chain Seq ID No: 84548-01 CDR1 Heavy Chain Seq ID No: 85 548-01 CDR2 Heavy Chain Seq ID No:86 548-01 CDR3 Heavy Chain Seq ID No: 87 548-01 VL Seq ID No: 88 548-01VH Ab 548-02 Seq ID No: 89 548-02 CDR1 Light Chain Seq ID No: 90 548-02CDR2 Light Chain Seq ID No: 91 548-02 CDR3 Light Chain Seq ID No: 92548-02 CDR1 Heavy Chain Seq ID No: 93 548-02 CDR2 Heavy Chain Seq ID No:94 548-02 CDR3 Heavy Chain Seq ID No: 95 548-02 VL Seq ID No: 96 548-02VH Ab1289-02 Seq ID No: 97 1289-02 CDR1 Light Chain Seq ID No: 981289-02 CDR2 Light Chain Seq ID No: 99 1289-02 CDR3 Light Chain Seq IDNo: 100 1289-02 CDR1 Heavy Chain Seq ID No: 101 1289-02 CDR2 Heavy ChainSeq ID No: 102 1289-02 CDR3 Heavy Chain Seq ID No: 103 1289-02 VL Seq IDNo: 104 1289-02 VH Ab 811-02 Seq ID No: 105 811-02 CDR1 Light Chain SeqID No: 106 811-02 CDR2 Light Chain Seq ID No: 107 811-02 CDR3 LightChain Seq ID No: 108 811-02 CDR1 Heavy Chain Seq ID No: 109 811-02 CDR2Heavy Chain Seq ID No: 110 811-02 CDR3 Heavy Chain Seq ID No: 111 811-02VL Seq ID No: 112 811-02 VH Ab 566-01 Seq ID No: 113 566-01 CDR1 LightChain Seq ID No: 114 566-01 CDR2 Light Chain Seq ID No: 115 566-01 CDR3Light Chain Seq ID No: 116 566-01 CDR1 Heavy Chain Seq ID No: 117 566-01CDR2 Heavy Chain Seq ID No: 118 566-01 CDR3 Heavy Chain Seq ID No: 119566-01 VL Seq ID No: 120 566-01 VH Ab 562 Seq ID No: 121 562 CDR1 LightChain Seq ID No: 122 562 CDR2 Light Chain Seq ID No: 123 562 CDR3 LightChain Seq ID No: 124 562 CDR1 Heavy Chain Seq ID No: 125 562 CDR2 HeavyChain Seq ID No: 126 562 CDR3 Heavy Chain Seq ID No: 127 562 VL Seq IDNo: 128 562 VH Ab 193 Seq ID No: 129 193 CDR1 Light Chain Seq ID No: 130193 CDR2 Light Chain Seq ID No: 131 193 CDR3 Light Chain Seq ID No: 132193 CDR1 Heavy Chain Seq ID No: 133 193 CDR2 Heavy Chain Seq ID No: 134193 CDR3 Heavy Chain Seq ID No: 135 193 VL Seq ID No: 136 193 VH Ab 88Seq ID No: 137 88 CDR1 Light Chain Seq ID No: 138 88 CDR2 Light ChainSeq ID No: 139 88 CDR3 Light Chain Seq ID No: 140 88 CDR1 Heavy ChainSeq ID No: 141 88 CDR2 Heavy Chain Seq ID No: 142 88 CDR3 Heavy ChainSeq ID No: 143 88 VL Seq ID No: 144 88 VH Ab 045 Seq ID No: 145 045 CDR1Light Chain Seq ID No: 146 045 CDR2 Light Chain Seq ID No: 147 045 CDR3Light Chain Seq ID No: 148 045 CDR1 Heavy Chain Seq ID No: 149 045 CDR2Heavy Chain Seq ID No: 150 045 CDR3 Heavy Chain Seq ID No: 151 045 VLSeq ID No: 152 045 VH Ab 044 Seq ID No: 153 044 CDR1 Light Chain Seq IDNo: 154 044 CDR2 Light Chain Seq ID No: 155 044 CDR3 Light Chain Seq IDNo: 156 044 CDR1 Heavy Chain Seq ID No: 157 044 CDR2 Heavy Chain Seq IDNo: 158 044 CDR3 Heavy Chain Seq ID No: 159 044 VL Seq ID No: 160 044 VHAb 002 Seq ID No: 161 002 CDR1 Light Chain Seq ID No: 162 002 CDR2 LightChain Seq ID No: 163 002 CDR3 Light Chain Seq ID No: 164 002 CDR1 HeavyChain Seq ID No: 165 002 CDR2 Heavy Chain Seq ID No: 166 002 CDR3 HeavyChain Seq ID No: 167 002 VL Seq ID No: 168 002 VH Seq ID No: 169 Fullhuman Sortilin sequence isoform 1 Seq ID No: 170 “D Region” asidentified by present invention Seq ID No: 171 Sortilin “hSORTECDBAP”Seq ID No: 172 Sortilin SORTECDBAP_hBACK Seq ID No: 173 SortilinSORTECDBAP_tetra Seq ID No: 174 Sortilin SORTECDBAP_hB01-05 Seq ID No:175 Sortilin SORTECDBAP_hRIM Seq ID No: 176 Sortilin SORTECDBAP_hB06-10Seq ID No: 177 Sortilin SORTECDBAP_hB12390 Seq ID No: 178 SortilinSORTECDBAP_hB45678 Seq ID No: 179 Sortilin SORTECD_HIS Seq ID No: 180 “Aregion” as identified by the present invention Seq ID No: 181 A region109-114 Seq ID No: 182 A region 126-153 Seq ID No: 183 A region 126-144Seq ID No: 184 A region 154-159 Seq ID No: 185 D Region 570-572 Seq IDNo: 186 D Region 588-597 Seq ID No: 187 D Region 593-597 Seq ID No: 188Sequences used for HDX

The listing or discussion in this specification of an apparentlyprior-published document should not necessarily be taken as anacknowledgement that the document is part of the state of the art or iscommon general knowledge.

EMBODIMENTS

As would be apparent from the text and the Examples the inventionfurther relates to the below embodiments:

-   1. An antibody, or an antigen-binding fragment thereof, capable of    specifically binding to Sortilin and inhibiting binding of PGRN to    Sortilin.-   2. The antibody, or antigen-binding fragment thereof, according to    Embodiment 1, wherein the antibody comprises or consists of an    intact antibody.-   3. The antibody, or antigen-binding fragment thereof, according to    Embodiment 1 or 2, wherein the antigen-binding fragment comprises or    consists of an antigen-binding fragment selected from the group    consisting of: an Fv fragment (e.g. single chain Fv or a    disulphide-bonded Fv); a Fab-like fragment (e.g. Fab fragment or    F(ab′)₂ fragment); and a domain antibody (e.g. a single VH variable    domain or VL variable domain).-   4. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, wherein the antibody is selected from the    group consisting of: an antibody of subtype IgG1, IgG2, IgG3 or    IgG4.-   5. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, wherein said antibody or antigen-binding    fragment thereof binds specifically to the D Region of Sortilin as    defined in SEQ ID NO:170.-   6. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, wherein said antibody or fragment thereof    binds specifically to at least 3 consecutive amino acids, such as 4,    5, 6 or 7 consecutive amino acids, of the D Region of Sortilin as    defined in SEQ ID NO:170.-   7. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, wherein the antibody or antigen-binding    fragment exhibits one or more of the following properties:    -   (i) a binding affinity (K_(D)) for Sortilin of between 0.5-10        nM, such as 1-5 nM or 1-2 nM    -   (ii) capability to reduce and/or inhibit PGRN binding to        Sortilin;    -   (iii) capability to reduce and/or inhibit clearance of PGRN by        Sortilin-expressing cells;    -   (iv) capability to reduce and/or inhibit the endocytosis of PGRN        by Sortilin-expressing cells;    -   (v) capability to increase the amount and/or concentration of        PGRN in the plasma in human-Sortilin-expressing knock-in mice.-   8. The antibody, or antigen-binding fragment thereof, according to    Embodiment 7, wherein the capability of the antibody or fragment    thereof to reduce PGRN binding to Sortilin comprises reducing PGRN    binding to Sortilin by 10% or more; for example, by 20% or more; or    by 30% or more.-   9. The antibody, or antigen-binding fragment thereof, according to    Embodiment 7 or 8, wherein the capability of said antibody or    fragment thereof the antibody or fragment thereof to reduce and/or    inhibit PGRN binding to Sortilin comprises reducing and/or    inhibiting PGRN binding to Sortilin with an IC50 at or below 22 nM,    such as between 22 nM and 1 nM, or between 10 nM and 1 nM, or    between 5 nM and 1 nM.-   10. The antibody, or antigen-binding fragment thereof, according to    any previous Embodiment, wherein the antibody or antigen-binding    fragment thereof is human or is humanized.-   11. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, comprising a light chain variable domain    comprising one or more of the CDR 1-3 Light Chain as listed for each    of the antibodies defined in Table 5, or an amino acid sequence    having no more than 4 amino acid differences, or no more than 3    amino acid differences, or no more than 2 amino acid differences, or    no more than 1 amino acid difference.-   12. The antibody, or antigen-binding fragment thereof, according to    Embodiment 11, comprising a light chain variable domain comprising    the CDR 1-3 Light Chain as listed for each of the antibodies defined    in Table 5.-   13. The antibody, or antigen-binding fragment thereof, according to    Embodiment 11 or 12, comprising a light chain variable domain    comprising or consisting of the amino acid sequence VL as listed for    each of the antibodies defined in Table 5.-   14. The antibody, or antigen-binding fragment thereof, according to    any of Embodiments 11 to 13, comprising a light chain comprising or    consisting of the amino acid sequence of VL as listed for each of    the antibodies defined in Table 5.-   15. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, comprising a heavy chain variable domain    comprising one or more CDR 1-3 Heavy Chain as listed for each of the    antibodies defined in Table 5, or an amino acid sequence having no    more than 4 amino acid differences, or no more than 3 amino acid    differences, or no more than 2 amino acid differences, or no more    than 1 amino acid difference.-   16. The antibody, or antigen-binding fragment thereof, according to    Embodiment 15, comprising a heavy chain variable domain comprising    the CDR 1-3 Heavy Chain as listed for each of the antibodies defined    in Table 5.-   17. An antibody, or antigen-binding fragment thereof, according to    Embodiment 15 or 16 comprising a heavy chain variable domain    comprising or consisting of the amino acid sequence of VH as listed    for each of the antibodies defined in Table 5.-   18. The antibody, or antigen-binding fragment thereof, according to    any of Embodiments 15 to 17, comprising a heavy chain comprising or    consisting of the amino acid sequence VL as listed for each of the    antibodies defined in Table 5.-   19. The antibody, or antigen-binding fragment thereof, according to    any preceding embodiment, comprising a light chain variable domain    comprising or consisting of the amino acid sequence of VL as listed    for each of the antibodies defined in Table 5, and a heavy chain    variable domain comprising or consisting of the amino acid sequence    of VH as listed for each of the antibodies defined in Table 5.-   20. The antibody, or antigen-binding fragment thereof, according to    any preceding embodiment, comprising a light chain comprising or    consisting of the amino acid sequence of VL as listed for each of    the antibodies defined in Table 5, and a heavy chain comprising or    consisting of the amino acid sequence of VH as listed for each of    the antibodies defined in Table 5.-   21. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, wherein said antibody or antigen-binding    fragment thereof competes with the antibody or antigen-binding    fragment thereof defined in Embodiment 20 for binding to Sortilin.-   22. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, wherein the antibody or antigen-binding    fragment comprises an Fc region.-   23. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, wherein the antibody or antigen-binding    fragment further comprises a moiety for increasing in vivo    half-life.-   24. The antibody, or antigen-binding fragment thereof, according to    Embodiment 22, wherein the moiety for increasing the in vivo    half-life is selected from the group consisting of polyethylene    glycol (PEG), human serum albumin, glycosylation groups, fatty acids    and dextran.-   25. The antibody, or antigen-binding fragment thereof, according to    any preceding Embodiment, wherein the antibody or antigen-binding    fragment further comprises a detectable moiety.-   26. The antibody, or antigen-binding fragment thereof, according to    Embodiment 25, wherein the detectable moiety is selected from the    group consisting of: a fluorescent label; a chemiluminescent label;    a paramagnetic label; a radio-isotopic label; or an enzyme label.-   27. The antibody, or antigen-binding fragment thereof, according to    Embodiment 25 or 26, wherein the detectable moiety comprises or    consists of a radioisotope.-   28. The antibody, or antigen-binding fragment thereof, according to    Embodiment 26 or 27, wherein the radioisotope is selected from the    group consisting of ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ¹²³I    and ²⁰¹TI.-   29. The antibody, or antigen-binding fragment thereof, according to    Embodiment 25, wherein the detectable moiety comprises or consists    of a paramagnetic isotope.-   30. The antibody, or antigen-binding fragment thereof, according to    Embodiment 29 wherein the paramagnetic isotope is selected from the    group consisting of ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr and ⁵⁶Fe.-   31. The antibody, or antigen-binding fragment thereof, according to    any of Embodiments 25 to 30, wherein the detectable moiety is    detectable by an imaging technique such as SPECT, PET, MRI, optical    or ultrasound imaging.-   32. The antibody, or antigen-binding fragment thereof, according to    any of Embodiments 25 to 31, wherein the detectable moiety is joined    to the antibody or antigen-binding fragment thereof indirectly, via    a linking moiety.-   33. The antibody, or antigen-binding fragment thereof, according to    Embodiment 32 wherein the linking moiety is selected from the group    consisting of: derivatives of    1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA);    deferoxamine (DFO); derivatives of diethylenetriaminepentaacetic    avid (DTPA); derivatives of    S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic    acid (NOTA); and derivatives of    1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA).-   34. An isolated nucleic acid molecule encoding an antibody or    antigen-binding fragment thereof as defined in any of Embodiments    1-33.-   35. A nucleic acid molecule according to Embodiment 34 wherein the    molecule is a cDNA molecule.-   36. A vector comprising a nucleic acid molecule as defined in    Embodiment 34 or 35.-   37. A recombinant host cell comprising a nucleic acid molecule as    defined in any of Embodiments 34-36.-   38. A method for producing an antibody or antigen-binding fragment    as defined in any of Embodiments 1-33, the method comprising    culturing a host cell as defined in Embodiment 37 under conditions    which permit expression of the encoded antibody or antigen-binding    fragment thereof.-   39. A preparation comprising the antibody or antigen-binding    fragment thereof according to any one of the previous Embodiments,    wherein said preparation is substantially free of naturally-arising    antibodies that are either not capable of binding to Sortilin or    that do not materially alter an anti-Sortilin functionality of the    preparation, said functionality being selected from the group    consisting of:    -   (i) a binding affinity (K_(D)) for Sortilin;    -   (ii) a capability to reduce and/or inhibit PGRN binding to        Sortilin;    -   (iii) a capability to reduce and/or inhibit clearance of PGRN by        Sortilin-expressing cells;    -   (iv) a capability to reduce and/or inhibit the endocytosis of        PGRN by Sortilin-expressing cells;    -   (v) a capability to increase the amount and/or concentration of        PGRN in the plasma in human-Sortilin-expressing knock-in mice;        and/or    -   (vi) a capability, when administered chronically, to provide        treatment of frontotemporal dementia (FTD) and/or amyotrophic        lateral sclerosis (ALS).-   40. A preparation comprising the monoclonal antibody or    antigen-binding fragment thereof according to any one of the    previous Embodiments, wherein said monoclonal antibody possesses a    structural change in its amino acid sequence, relative to the    structure of a naturally-occurring anti-Sortilin antibody, wherein    said structural change causes said monoclonal antibody to exhibit an    altered functionality relative to the functionality exhibited by    said naturally-occurring anti-Sortilin antibody, wherein said    functionality is:    -   (i) a binding affinity (KD) for Sortilin;    -   (ii) a capability to reduce and/or inhibit PGRN binding to        Sortilin;    -   (iii) a capability to reduce and/or inhibit clearance of PGRN by        Sortilin-expressing cells;    -   (iv) a capability to reduce and/or inhibit the endocytosis of        PGRN by Sortilin-expressing cells;    -   (v) a capability to increase the amount and/or concentration of        PGRN in the plasma in human-Sortilin-expressing knock-in mice;        and/or    -   (vi) a capability, when administered chronically, to provide        treatment of frontotemporal dementia (FTD) and/or amyotrophic        lateral sclerosis (ALS).-   41. A pharmaceutical composition comprising an antibody, or    antigen-binding fragment thereof, as defined in any of Embodiments    1-33, or the preparation of any one of embodiments 39-40, and a    pharmaceutically-acceptable carrier.-   42. The antibody, or antigen-binding fragment thereof, as defined in    any of Embodiments 1-33, or the preparation of any one of    embodiments 39-40, for use in medicine.-   43. The antibody, or antigen-binding fragment thereof, as defined in    any of Embodiments 1-33, or the preparation of any one of    embodiments 39-40, for use in preventing and/or treating a disease    associated with decreased PGRN levels in the brain of a patient.-   44. Use of an antibody, or antigen-binding fragment thereof, as    defined in any of Embodiments 1-33, or the preparation of any one of    embodiments 39-40, in the manufacture of a medicament for preventing    and/or treating a disease associated with decreased PGRN levels in    the brain of a patient.-   45. The antibody or antigen-binding fragment thereof for use    according to Embodiment 43, or the use according to Embodiment 44,    wherein the disease is selected from the group consisting of: FTD;    ALS; TDP43 proteinopathies, such as AD.-   46. A method of preventing or treating a disease associated with    decreased PGRN levels in the brain of a patient, comprising    administering an effective dosage of an antibody or a fragment    thereof as defined in any of Embodiments 1-33, the preparation of    any one of Embodiments 39-40, or the pharmaceutical composition of    Embodiment 41.-   47. The antibody, or antigen-binding fragment thereof, for use    according to Embodiment 43, or the use according to Embodiment 44,    or the method according to Embodiment 46, wherein the disease is    selected from the group consisting of: FTD; ALS; or TDP43    proteinopathies, such as AD.-   48. The antibody, or antigen-binding fragment thereof, for use; or    the use; or the method according to Embodiment 46 or 47, wherein the    treatment is chronic.-   49. The antibody, or antigen-binding fragment thereof, for use; or    the use; or the method, according to Embodiment 48, wherein the    chronic treatment is for at least 2 weeks, such as at least for 1    month, 6, months, 1 year or more-   50. The antibody, or antigen-binding fragment thereof, as defined in    any of Embodiments 1-33, the preparation of any one of Embodiments    39-40, or the pharmaceutical composition of Embodiment 41, which is    capable of specifically binding to Sortilin and inhibiting the    binding of PGRN to Sortilin, but which binding does not inhibit or    substantially inhibit the binding of neurotensin or AF38469 to    Sortilin.-   51. A kit comprising the antibody, or antigen-binding fragment    thereof, as defined in any of Embodiments 1-33, the preparation, as    defined in any one of Embodiments 39-40, or the pharmaceutical    composition as defined in Embodiment 41.

Preferred, non-limiting examples which embody certain aspects of theinvention will now be described, with reference to the accompanyingfigures.

EXAMPLES

Examples 1-3 describe the generation of sortilin constructs

Example 1 discloses the shuffle constructs. Example 2 discloses theexpression of sortilin constructs. Example 3 discloses the purificationof sortilin constructs.

Examples 4-7 describe the generation of sortilin antibodies

Example 4 discloses the immunization and the hybridomas.

Example 5 discloses the sequence analysis. Example 6 discloses thepurification of antibodies. Example 7 discloses the generation of mouseantibodies.

Examples 8-17 describe the characterization of sortilin antibodies

Example 8 discloses the binding to sortilin. Example 9 discloses thecross blocking ability of Sortilin antibodies. Example 10 discloses HTRFPGRN-sortilin binding. Example 11 discloses NTS binding. Example 12discloses cellular PGRN binding and endocytosis. Example 13 disclosesextracellular PGRN levels. Example 14 discloses iPSC PGRN levels.Example 15 discloses a plasma PGRN levels. Example 16 disclose epitopemapping by HDX.

Example 17 disclose microdialysis of PGRN in the brain.

Example 1

For use in both the hybridoma screening process and as a diversificationof the panel of antibodies, so called “shuffle constructs” weredesigned, constructed and produced, making a set of chimeric sortilinmolecules containing amino acid sequences derived from both humansortilin and a distantly related species (tetraodon) with significantlyreduced sequence homology. The rationale being that the overall sortilinstructure and functionality of these chimeric constructs would beretained but that loss of binding of antibodies to certain chimericconstructs would indicate the involvement of the specific exchangedregions in binding. Soluble extracellular region (ECD, aa 1-755)constructs were tagged with either a BAP tag (biotin acceptor peptide),enabling the “in vitro” biotinylation of the proteins by co-expressionof biotin ligase or a His tag, enabling easy purification. Expressionvectors encoding the following proteins were prepared: SORT-ECDBAP,SORT-ECDBAP-hB01-05, SORT-ECDBAP-hB06-10, SORT-ECDBAP-hB12390,SORT-ECDBAP-hB45678, SORT-ECDBAP-tetra, SORT, SORT-tetra.

The Sortilin sequences can be found in SEQ ID NOs:169-180 and FIGS.2A-2C show schematic presentation of the region assignment of antibodiesbased on binding to Sortilin shuffle constructs.

Example 2

In the case of antibody expression, the appropriate heavy chain andlight chain vectors, as described in Examples 4, 5 and 6, wereco-expressed in HEK-293F cells.

Example 3: Purification of His-Tagged Sortilin

SORTECDHis was expressed in HEK-293F cells. The His-tag in the proteinsenables purification with immobilized metal affinity chromatography. Inthis process NiNTA Superflow Cartridge (Qiagen) is equilibrated with 50mM NAH₂PO₄, 300 mM NaCl and 10 mM Imidazole pH 8.0. Column is loadedwith His tagged protein with a residence time of 1 minute. Column iswashed with 50 mM NAH₂PO₄, 300 mM NaCl and 20 mM Imidazole pH 8.0.Protein is eluted with 50 mM NAH₂PO₄, 300 mM NaCl and 250 mM ImidazolepH 8.0. Subsequently the protein is dialyzed to PBS using aSlide-A-Lyzer with a cut off of 10.000 mwco (Thermo Scientific). Afterdialyzing the protein is sterile filtered using a 0.2 micron SFCAfilter(Thermo Scientific).

The S18-HEK cell line was generated by transfecting HEK293 cells with ahuman wild type (WT) sortilin expression vector. Stable transfectedcells were derived after passage in the presence of a selection agent.Individual clones were selected by dilution cloning. Clones werecharacterized for sortilin mRNA expression using QPCR. Highestexpressing clones were than analyzed by FACS (Guava, Millipore) using ananti-sortilin polyclonal antibody (Polyclonal Goat Sortilin BiotinylatedAb, Cat. No: BAF2934_(R&D Systems)) to determine the surface expressedlevels of Sortilin.

Example 4

A—Immunization Procedure of Transgenic Mice

Antibodies HuMab Sortilin were derived from the immunizations of HuMAbmouse strains HCo12, HCo17, HCo20, HCo12-BALB/c, HCo17-BALB/c andHCo20-BALB/c (human monoclonal antibody; Medarex Inc., San Jose, Calif.,USA), These mice are double knock out for the mouse immunoglobulin (Ig)heavy and mouse kappa light chain, which substantially inactivate theexpression of antibodies that are completely murine. The various mousestrains were made transgenic by the insertion of human Ig heavy andhuman Ig kappa light chain loci and differ in the number of human VH(variable domain of heavy chain) and VL (variable domain of light chain)genes. HCo12-BALB/c mice were derived by crossbreeding with KCo5-BALB/c(kappa light chain transgenic) mice.

48 mice were immunized alternating intraperitoneally (IP) with 20 μgSORTECDHis (SEQ ID NO: 179) and subcutaneously (SC, at the tail base)with the same protein, with an interval of 14 days. A maximum of eightimmunizations were performed, 4 IP and 4 SC.

In one protocol, the first immunization was performed with SORTECDHis incomplete Freund's adjuvant (CFA; Difco Laboratories, Detroit, Mich.,USA), the following immunizations in incomplete Freund's adjuvant (IFA).A second protocol used SAS as an adjuvant in all immunization steps.When serum titers were found to be sufficient (dilution of serum of 1/50or lower found positive in antigen specific screening assay on at leasttwo sequential, biweekly, screening events), mice were additionallyboosted twice intravenously (IV) with 10 μg SORTECDHis protein in 100 μLPBS, four and three days before fusion.

B—HuMab Hybridoma-Generation

HuMAb mice with sufficient antigen-specific titer development as definedabove were sacrificed and the spleen and lymph nodes flanking theabdominal aorta and caval vein were collected. Fusion of splenocytes andlymph node cells with a mouse myeloma cell line was done byelectrofusion using a CEEF 50 Electrofusion System (Cyto Pulse Sciences,Glen Burnie, Md., USA), essentially according to the manufacturer'sinstructions. Fused cells were seeded in fusion medium containing 10%Fetal Clone I Bovine serum (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5U/mL penicillin, 0.5 U/mL streptomycin (Cambrex), 50 μM2-mercaptoethanol (Invitrogen), 600 ng/mL interleukin 6 (IL-6)(Strathmann), 1×HAT (Sigma) and 0.5 mg/mL kanamycin (Invitrogen) in HyQmADCF-Mab (Perbio). After ten days, supernatant was harvested and cellswere refreshed with harvest medium, containing 10% Fetal Clone I Bovineserum, 0.5 U/mL penicillin, 0.5 U/mL streptomycin, 600 ng/mL IL-6 and1×proHT (Cambrex) in HyQ mADCF-Mab. Supernatants of the hybridomacultures were screened by primary screening assays and streptavidinbeads coupled to SORTECDBAP (SEQ ID NO 171), SORTECDBAPhB06-10 (SEQ IDNO 176), SORTECDBAPhB12390 (SEQ ID NO 177), to detect hybridomasproducing human (or chimeric) anti-Sortilin antibodies. Hybridoma cellsfrom the best primary wells were seeded in semisolid medium made from40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2× complete medium(Hyclone, Waltham, USA). For each primary well, a well of a Genetixblack 6-well plate was seeded. From each well, 25 sub clones werepicked, using the ClonePix system (Genetix). The sub clones were pickedin harvest medium. After seven days, the supernatants of the sub cloneswere screened again for Sortilin-specific human IgG binding and thehuman IgG concentration was measured using Octet 384red (Fortebio, MenloPark, USA). From each primary well, the best sub clone was selected andexpanded in expansion medium containing only 600 ng/mL IL-6, 0.5 U/mLpenicillin, 0.5 U/mL streptomycin and 1×proHT. The sub clones wereexpanded from one 96-well plate well to one 24-well plate well to four24-well plate wells to six 6-well plate wells. Clones derived by thisprocess were designated as primary clones (PC).

The anti-sortilin HuMab antibodies of the invention were identified andsubjected to sequence analysis.

Example 5: Sequence Analysis of the Sortilin-Specific HuMab VariableDomains and Cloning in Expression Vectors

Total RNA was prepared from 0.2 to 5×106 hybridoma cells and5′-RACE-Complementary DNA (cDNA) was prepared from 100 ng total RNA,using the SMART RACE cDNA Amplification kit (Clontech), according to themanufacturer's instructions. VH and VL coding regions were amplified byPCR and cloned directly, in frame, in the p33G1f and p33Kappa expressionvectors (containing the human IgG1./kappa constant domain encodingsequences), by ligation independent cloning (Aslanidis, C. and P. J. deJong, Nucleic Acids Res 1990; 18(20): 6069-74). For each antibody, 16 VLclones and 16 VH clones were sequenced. Clones with a correct OpenReading Frame (ORF) were selected for further study and expression.Vectors of all combinations of heavy chains and light chains weretransiently co-expressed in Freestyle™ 293-F cells using 293fectin.

The resulting sequences are shown in the Sequence Listing (SEQ IDNOs:1-168) herein. CDR sequences were defined according to the publishedguidelines.

Example 6: Purification of Antibodies

Culture supernatant was filtered over 0.2 μm dead-end filters, loaded on5 mL Protein A columns (rProtein A FF, Amersham Bioscience) and elutedwith 0.1 M citric acid-NaOH, pH 3. The eluate was immediatelyneutralized with 2M Tris-HCl, pH 9 and dialyzed to 12.6 mM NaH₂PO₄, 140mM NaCl, pH 7.4 (B. Braun), O/N (over night). After dialysis, sampleswere sterile-filtered over 0.2 μm dead-end filters. Purity wasdetermined by SDS-PAGE and concentration was measured by nephelometryand absorbance at 280 nm. Purified antibodies were aliquoted and storedat −80° C. Once thawed, purified antibody aliquots were kept at 4° C.Mass spectrometry was performed to identify the molecular mass of theantibody heavy and light chains expressed by the hybridomas.

Example 7: Generation of Mouse Antibodies (1F2 and 5E1) Immunogen

A synthetic gene coding for the chimeric immunogen hSortilin-FC, (humanSortilin AA (78-756) from SEQ ID NO:169) and human IgG1-FC AA (104-330)from SEQ ID NO:169 was cloned into pcDNA3.1 and used for expressionusing the freestyle system from Invitrogen. The antigen was purifiedfrom cell culture supernatants by protein-A affinity chromatographyusing standard procedures for antibody purification as described abovefor human antibodies.

Hybridoma Generation

hSortilin-FC was used as immunogen and 5 BALB/c mice were immunized. Amouse with satisfactory immune response was selected for cell fusion andhybridoma generation. Hybridoma supernatants were screened by ELISAusing hSortilin-ECD as coating antigen. A total of eighteen hybridomacell lines derived from nine parental clones were generated.

Expression

Hybridomas were initially grown in complete growth medium, DMEM with 10%FBS+ antibiotics, and subsequently adapted to CDhybridoma media(Invitrogen) for expression experiments.

Purification

Mouse monoclonal antibodies were purified from hybridoma cell culturesupernatants by protein-G sepharose according to standard proceduresrecommended by the supplier (GE healthcare).

Example 8: Affinity of Sortilin Specific HuMab and Mouse Antibodies toRecombinant Extracellular Region of Sortilin

Binding kinetics of anti-Sortilin HuMab antibodies to Sortilin weredetermined using Octet 384RED (Fortebio, Menlo Park, USA). HuMabsolutions of 2 μg/ml were made by dilution in sample diluent (ForteBio,art. No. 18-5028). Prot A sensors (ForteBio, art.no. 18-0004) wereprewetted with kinetics buffer (1:10 sample diluent in PBS) for at least600 seconds. Subsequently sensors were immobilized with HuMab solutionfor 600 seconds. A baseline response was obtained by dipping in kineticsbuffer for 120 seconds. Association of SORTECD constructs was performedduring a 1000 seconds incubation. This was followed by dissociation inkinetics buffer for 100 seconds. After dissociation, sensors wereregenerated (10 mM Glycine pH 1.0) and neutralized (kinetics buffer) 3times for 5 seconds. All HuMab were analysed using four concentrationsof SORTECD constructs (10, 5, 2.5 and 1.25 μg/ml). A molecular weight of76.8 kDA was used for SORTECDHis. Data was fitted with ForteBio Analysis6.4 software, using a global full fit. Results are shown in FIG. 3 andFIG. 4 .

Example 9: Antibody Cross Block of Anti-Sortilin HuMabs

Antibody cross-block studies were performed using Octet 384RED(Fortebio, Menlo Park, USA). HuMab antibody solutions of 2 μg/ml weremade by dilution in sample diluent (ForteBio, art. No. 18-5028). Aminereactive sensors (ForteBio, art.no. 18-0008) were used forimmobilization of HuMabs. Prior to coupling to amine reactive sensors,HuMabs were diluted in MES pH 6.0 buffer (18-5027). Coupling wasperformed at 30° C. and 1000 rpm as follows: Amine reactive sensors wereprewet in PBS and subsequently activated with EDC/NHS (ForteBio. Art.no.18-1033/18-1034) activation solution (according to manufacturer'sinstruction) for 300 seconds. Activated sensors were immobilized withHuMabs during 600 seconds. Immobilized sensors were quenched forremaining amine reactivity with Ethanolamine (ForteBio, cat no.18-1039). After quenching sensors were placed in PBS until use. Crossblock analysis starts with establishing a baseline response at 30° C.and 1000 rpm. Baseline response was obtained by dipping in samplediluent for 120 seconds. Association of SORTECDHis was performed during300 seconds directly followed by association of HuMab for 300 seconds.After association of HuMab, sensors were regenerated (10 mM Glycine pH1.0) and neutralized (sample diluent) 3 times for 5 seconds. Data wasprocessed using ForteBio Analysis 6.4 software.

Antibodies were grouped based on their binding profiles on the differentSortilin shuffle constructs (FIGS. 2A-2C, FIG. 3 and FIG. 4 ). Toconfirm that all the antibodies from Region D (and region F) bind to thesame region on human wild type Sortilin ECD, their ability to block eachother's binding to the wild type human Sortilin ECD was characterised ina cross blocking study using the Octet384 red. For example, whenantibodies from the same region were tested, the primary antibody wouldblock binding of the secondary antibody and vice versa. Whereas, whenantibodies from different regions were tested, there would be no crossblocking as only one region is blocked by the primary antibody and theremaining regions are available for the secondary antibody to bind.FIGS. 7A-7C show that all D-region and D+ antibodies cross block eachother which confirms classification of the antibodies to Region D and D+based on shuffle constructs. Further, these data also confirm that thechimeric Sortilin constructs retain similarity to the native human wildtype Sortilin ECD.

Example 10: Characterization of Sortilin—PGRN Ligand Binding in thePresence of Anti-Sortilin Antibodies

IC50 values for antibodies were determined by measuring the displacementof PGRN binding to Sortilin using a homogenous time resolved fluorescent(HTRF, CisBio) assay, see FIG. 5 and FIG. 6 .

Experiments were performed in assay buffer (50 mM Phosphate, pH 7.0,0.1% BSA) in a total volume of 20 μl in a Greiner 384 well, white, lowvolume microtiter plate, (784075, Greiner).

The antibodies were pre-incubated for 15 min at room temperature with 50nM HIS-tagged Sortilin ECD and 4 nM PGRN (SULU20110924) before 7 nManti-6HIS-d2 and 0.7 nM anti-PGRN-Eu cryptate (Cisbio) diluted inconjugate buffer (50 mM Phosphate, pH 7.0, 0.8 mM KF, 0.1% BSA) wereadded. 20 μM neurotensin was used as positive control and DMSO in bufferwas used as negative control.

The assay plate was incubated for 60 min at room temperature andovernight at 4° C. before the plate was read in EnVision reader (PerkinElmer).

Unlabelled neurotensin and DMSO blank were used as positive and negativecontrols for the assay set up, respectively. Dose-response evaluation ofantibodies was performed with ten concentrations between 1 μM and 50 μMin a 3-fold dilution curve.

The half-maximal inhibitory concentration (IC50) were calculated bynon-linear regression using a sigmoidal concentration response (variableslope) in XLfit 4 (IDBS, UK). (FIGS. 5 and 6 ).

Example 11: Characterization of Sortilin—Neurotensin Binding in thePresence of Anti-Sortilin Antibodies

The IC50 for the Sortilin specific compound AF38469 (Schroder et al,Bioorg Med Chem Lett. 2014 Jan. 1; 24(1):177-80, 2014) was determined bymeasuring the displacement of ³H-Neurotensin binding to Sortilin using ascintillation proximity assay (SPA).

Experiments were performed in assay buffer (50 mM HEPES, pH 7.4, 100 mMNaCl, 2 mM CaCl₂), 0.1% BSA, 0.1% Tween-20) in a total volume of 40 μlin a 384-well Optiplate, white opaque, (6007299, Perkin Elmer).

150 nM HIS-tagged Sortilin was pre-incubated for 15 min at roomtemperature with or without 1 μM Sortilin specific antibody(IgG1-6003-045 or IgG1-6003-068) or with human IgG1 isotype controlbefore the protein solutions were added to wells containing AF38469 in aconcentration series 50 μM to 2.5 nM. The mixture was incubated for 15min at room temperature on a shaker before 5 nM ³H-Neurotensin andNi-chelate imaging beads (RPNQ0266, Perkin Elmer) were added. The assaywas incubated additionally for 60 min under same conditions.

After 6 h, the plate was read on ViewLux (360 s exposure time).Unlabelled neurotensin and DMSO blank were used as positive and negativecontrols, respectively.

Dose-response evaluation for AF38469 was performed with tenconcentrations between 50 μM and 2.5 nM in a 3-fold dilution curve. Thehalf-maximal inhibitory concentration (IC50) were calculated bynon-linear regression using sigmoidalconcentration response (variableslope) in XLfit 4 (IDBS, UK). Results can be seen in FIG. 8 .

Example 12: Characterization of Sortilin—PGRN Ligand Binding on theSurface of Cells in the Presence of Anti-Sortilin Antibodies

Both Sortilin transiently transfected cells and the stable cell lineS18-HEK cells (human Sortilin over-expressing HEK cells) were used inthis assay. Cells were trypsinized and plated at density of 42,000 cellsper well in a 96 well plate. In the case of transiently transfectedcells, the cells were plated 24 hrs after transfection in 96 wellplates. Next day, media was changed completely and test compoundsdiluted in media were added to cells for 30 min. followed by addition ofPGRN for 4 hr. At the end of the study (after 4.5 hrs), the cells werefixed and stained for PGRN. All stained plates were analyzed byCellomics Array Scan (Thermo Fischer) and average staining intensity forPGRN/cell/well was used for analysis.

PGRN used in the assay was harvested from media following transienttransfection of PGRN expression plasmids in HEK 293 cells. PGRN levelswere measured using PGRN ELISA kit (R&D).

PGRN added to cells was readily bound and endocytosed which led to anincreased fluorescence signal in Sortilin transfected wells. Addition ofneurotensin, prevented Sortilin binding to PGRN and a PGRN fluorescenceintensity is similar to control levels indicating that PGRN was notbound and endocytosed in the presence of neurotensin.

Both Sortilin HumAbs (45 and 68) blocked the uptake of PGRN with anefficacy similar to neurotensin. The isotype control antibody, B12, didnot have any effect on PGRN endocytosis or binding. Results can be seenin FIG. 9 .

Example 13: Effect of Antibodies on Extracellular PGRN Levels

Both HEK293 cells and S18-HEK cells were found to secrete PGRNcontinuously into the medium without any stimulation.

Antibodies and control agents were added to S18-HEK cells to evaluatethe effect on PGRN. Addition of Neurotensin, a known peptide sortilinligand, or human antibodies, 45, 68 and 811 to S18-HEK cells led to anincrease of PGRN in the cell culture medium. Two of the Sortilin humanantibodies (45 and 68) had an effect similar to neurotensin elevatingPGRN levels to 202% and 201% respectively. The antibody 811 increasedPGRN to 146% in the medium as compared to the controls B12, an isotypecontrol antibody was used as negative control in all our studies and didnot show any effect on PGRN levels. These observations indicate that thetested sortilin antibodies inhibited sortilin-mediated internalizationof PGRN, thereby increasing extracellular PGRN.

On day1, S18-HEK cells were seeded in a 96 well plate. After 24 hrs,medium was completely replaced with either medium alone (control) ormedium supplemented with test compound. All compounds were tested at 10uM and antibodies at 100 nM unless otherwise specified. Medium wascollected on day 3 and analyzed using PGRN ELISA (R&D). Cell viabilitywas assessed by Cell TiterGlo (Pro Mega) to evaluate cytotoxic effect ofthe compounds. PGRN levels in media were analysed by ELISA and valueswere normalized to control wells. Results can be seen in FIG. 10 .

Example 14: ELISA Assay for Extracellular PGRN in iPSCs

Induced pluripotent stem cells (iPSCs) were generated by non-integrativereprogramming of human fibroblasts (Normal human dermal fibroblasts 18 ymale; Lonza) as described elsewhere (Rasmussen et al., Stem CellReports. 2014 Sep. 9; 3(3):404-13.). The NHDF K1_shp53 line was used forthese studies. The iPSCs were initially generated in mTESR media andsubsequently cultured in monolayer in Pluripro (Cell Guidance System).Neuronal differentiation was initiated day 0 by replating the cells onpoly-L-ornithine/laminin coated dishes and culturing them in N3 media(50% DMEM/F12+50% Neurobasal media supplemented with 0.5% N2, 1% B27with RA, 0.5 mM GlutaMAX, 0.5& NEA, 50 μM 2-mercaptoethanol and 2.5mg/mL insulin) with 500 ng/mL noggin and 10 μM SB431542. The media wasrefreshed every day. After 11 days of noggin/SB431542 induction, thecells were split with dispase and re-plated on poly-L-ornithine/lamininin N3 media. From that point forward, N3 media was refreshed every 2-3days and the cells were split approximately every 10-14 day usingaccutase.

Neuronal differentiated iPSC cells were plated into 96 wells plate. Oneweek later, antibodies were added to the cells. Media from the cellswere collected at 48 hrs or 96 hrs and analysed by human PGRN ELISA(Enzo Life sciences) and samples analysed as per the manufacturer'sinstructions.

The tested Sortilin human antibodies (45 and 68) increased PGRN levelsat varying levels in the media at 48 hrs and 96 hrs. B12 and Anti-Helare the control isotype antibodies (negative control). Data is presentedas mean±SD. Data was analyzed by one-way Anova followed by Dunnett'sanalysis *p<0.05; **p<0.01. Results can be seen in FIG. 12 .

Example 15

To analyze the effect of antibodies on PGRN levels in plasma, humanizedSortilin KI mice were given a single or multiple injections (10 mg/kg)of the sortilin antibodies or isotype control by subcutaneousinjections. The animals were anaesthetized and sacrificed at varioustime points after dosing and plasma PGRN levels determined by ELISA.

-   -   A. Time course study: Mice were treated with antibodies (a        Sortilin humab or a control ab) and sacrificed at different time        points. Mice treated with control antibodies (Anti-Hel), did not        show change in plasma PGRN whereas in mice treated with Sortilin        humab 45, there was an increase in PGRN levels which seemed to        peak between 24 and 48 hrs and then gradually decreased from        around day 4. PGRN levels were still elevated at day 7.    -   B. Subchronic study: Based on the data from time course study,        Sortilin KI mice were dosed twice a week with 10 mg/kg, s.c,        with either Sortilin human antibody 45 or isotype control        antibody, in order to maintain a steady antibody level for a        subchronic study (4 weeks). Blood samples were collected at the        start of the study and every week to follow plasma PGRN changes.        Plasma PGRN levels at the start of the study were similar in        both group of animals. Higher levels of plasma PGRN were seen in        mice treated with Sortilin antibody45 from week 1 and remain        elevated throughout the study. Mice treated with control ab, did        not show any increase in plasma PGRN and remained at baseline        levels (week 0).    -   C. Dose response study: Different doses (4 doses: 10, 2, 0.4 and        0.1 mg/kg) of the Sortilin (45) and control antibody (Anti-Hel)        were injected and mice sacrificed on day 2. Plasma PGRN was        elevated with 10 mg/kg and 2 mg/kg in mice treated with Sortilin        humab and the lower doses did not have an effect on the plasma        PGRN which clearly shows a dose dependent effect of the Sortilin        antibody on the plasma PGRN levels. Mice treated with control        antibody did not show any change in PGRN levels.

Mice were anaesthetized with 0.4 ml Avertin IP and heart blood wascollected and transferred to a 500 ul kEDTA vial. Samples were kept onice until centrifuged at 3600 G for 15 min at 4 C. The plasma waspipetted in to a micronic vial and frozen at −20 C. PGRN in the sampleswas measured using PGRN ELISA kit (Adipogen) as per the manufacturer'sinstructions. Results can be seen in FIGS. 13A-13C.

Example 16: Epitope Mapping of Antibodies Targeting theProgranulin-Sortilin Interaction by Hydrogen/Deuterium Exchange Followedby Mass Spectrometry

In hydrogen/deuterium exchange followed by mass spectrometry (HDX-MS)the exchange rate of backbone amide hydrogens in a protein is measured.Hereby, it is possible to probe the conformational dynamics of theentire protein backbone except at proline residues. The rate of theexchange reaction is determined by the hydrogen bonding status of thebackbone amide and to a lesser extent its solvent accessibility. Subtlechanges in these two parameters e.g. caused by the presence of a ligandcan be observed as a change in deuterium incorporation.

To sub-localize the changes in deuterium incorporation the protein istreated with an acid stable protease (e.g. pepsin), which generateslocal regions of typically ten to fifteen amino acids. Regions thatshows a perturbation in the presence of a ligand is either directlyinvolved in the binding interface or allosterically affected by thebinding event.

Epitope Mapping of Antibodies

The deuterium incorporation of the extra cellular region of Sortilin(SEQ ID NO:188) was measured in the absence and presence of mAb45,mAb68, mAb811 and an antibody denominated mAb30 which does not bind theD region. To secure that the measurements were conducted at steady-stateconditions the complexes were equilibrated for 15 min at 25° C. beforethe exchange reaction was initiated. The exchange reaction was initiatedby dilution of the protein samples 1:9 (v/v) into deuterated buffer (99%D2O, 20 mM tris, 150 mM NaCl, pDread=7.6). After various time points (15s, 1 min, 10 min, 1 h and 8 h) the exchange reaction was quenched by 1:1(v/v) dilution with ice-cold quench buffer (2M glycine, 0.8Mtris-(2-carboxyethyl)phosphine (TCEP), pH=2.3), thereby decreasing thepH to 2.46. The quenched samples were immediately placed inside a −80°freezer and stored until analysis. Fully deuterated control samples wereprepared by diluting sortilin samples 1:9 (v/v) into a deuterateddenaturation buffer (6M guanidinium chloride, 99% D20, 20 mM tris, 150mM NaCl, pDread=7.6) followed by incubation at 25° C. for 16 h beforethey were quenched and handled as described above.

The quenched samples were thawed and injected into a cooled (0° C.)reverse-phase UPLC-HDX-system (Waters Inc., USA) equipped with ahome-packed pepsin column (internal volume of 60 μL, pepsin beadsacquired from Thermo Scientific Inc.). Here, the deuterated proteinsamples were subjected to online pepsin digestion at 20° C., and theresulting peptic peptides were separated by reverse-phase UPLC. Thepeptides were ionized by electrospray ionization into a massspectrometer (Synapt G2 mass spectrometer, Waters Inc, UK), where thepeptides were further separated by ion mobility before final massdetermination.

The Identification of peptides was performed on fully reduced andnon-deuterated samples by tandem mass spectrometry using a combinationof data independent (MSe) and data dependent acquisition.

Data Analysis

Identification of Peptides

The acquired mass spectra were lock mass corrected against GFP andanalyzed in PLGS 3.0, which matched precursor and fragment ions to alocal protein database. All peptide identifications were carefullyassessed manually.

Determination of deuterium incorporation: The acquired mass spectra werelock mass corrected against GFP and the software DynamX 3.0 (WatersInc., USA) was used to determine the deuterium incorporation for allpeptides of sortilin either in absence or presence of antibodies.

A peptide was considered to be a part of the binding epitope if aprotection from exchange larger than 0.5D was observed in presence of anantibody.

TABLE 1 Table of identified conformational epitopes by HDX-MS. AntibodyEpitope mapping by HDX-MS relative to SEQ ID NO: 169 45 109-114 126-153570-572 588-597 68 109-114 126-144 154-159 570-572 593-597 811-02109-114 126-144 593-597

Example 17: Microdialysis to Assess Progranulin Levels in the Brain ofAwake Freely Moving Animals

Push-pull microdialysis method was used to assess brain ISF progranulin(PRGN) from awake and freely moving mice. Mice were single-housed incontrolled temperature (22±1.5° C.) and humidity conditions (55-65%) andkept in a 12:12 hour light/dark cycle (lights on at 06:00 h). Food andwater were available ad libitum. The current study was performed in thehippocampus of human sortilin knock-in (hSORT1) mice (22 weeks old). Toenable microdialysis in the hippocam-pus, mice were anaesthetized withisoflurane and an intracerebral guide cannula (CMA) was stereotaxicallyimplanted into the brain, positioning the microdialysis probe in thehippocampus (co-ordinates of probe tip: 3.1 mm posterior and 2.8 mmlateral from bregma, and 1.3 mm relative dura mater) according to theatlas of Paxinos and Franklin 2001. Acrylic cement was used for thefixation of the guide cannulas. After implantation of the cannula micewere allowed to recover from the surgery for 7 days before dialysis.During the first 5 days, including the surgery day, animals had pain andantibiotics treatments (Rimadyl and Noromox Prolongatum). 24 h beforethe starting of microdialysis experiments pump was also connected to theoutlet tubing in order to prevent perfusion fluid loss from the probe,by pulling the fluid through the tubing. As a perfusion buffer, 25%bovine albumin fraction V (Sigma) was diluted to 0.2% with artificialCSF (aCSF; in mM: 147 NaCl, 2.7 KCl, 1.2 CaCl2, 0.85 MgCl2) on the dayof use and filtered through a 0.1-μm membrane. The actual flow rate ofthe pump was determined without having the probe connected. The sampletubes were weighed before and after sampling for a given time period andthe flow rate was calculated. The pump was then set to have a constantflow of 1 μL/min. A 120-min sampling regimen was used throughout theexperiment period and 12 samples (12 h of collection) were collected(FIG. 17 , for procedure). At the end of experiments, blood was takenfrom animals, animals were perfused and brains collected. Thedialysates, plasma and brains were stored at −80° C. until PRGNdetermination by ELISA.

The measurement of PRGN levels every 2 h during 24 h is depicted in FIG.17 . At every time period, except 24 h after starting collecting didialysates, PRGN levels are significantly increased in animals-treatedwith mab #45 increased when compared to the ones from animals treatedwith PBS (FIG. 18A). PRGN levels are stable over time from 4 h until 16h after probe insertion in hippocampus. In the first dialysate PRGN areelevated likely due to the probe insertion into hippocampus. It isspeculated that PRGN levels are decreasing >18 h/20 h after probeinsertion, likely due to the clogging of the probe membrane, as itoccurred in both groups (and has been previously observed in otherpush-pull studies).

The average±SEM of the 12 dialysis samples 24 h after antibody orvehicle treatment, for each animal and then all animals pooled, wastaken as baseline (FIG. 18B). Differences between animals-treated withmab #45 and PBS were analyzed with unpaired t-test. The basal levels ofPRGN in animals-treated with mab #45 were significantly increased whencompared to the ones from animals treated with PBS (p<0.001, F10.0, DFn,9 Dfd 7; 3.3±0.3 ng/ml, n=10 versus 1.1±0.1 ng/ml, n=8) (FIG. 18B).

The invention claimed is:
 1. An antibody, or an antigen-binding fragmentthereof, comprising: (a) a light chain variable domain L-CDR1 comprisingSEQ ID NO:137; (b) a light chain variable domain L-CDR 2 comprising SEQID NO:138; (c) a light chain variable domain L-CDR 3 comprising SEQ IDNO:139; (d) a heavy chain variable domain H-CDR 1 comprising SEQ IDNO:140; (e) a heavy chain variable domain H-CDR 2 comprising SEQ IDNO:141; and (f) a heavy chain variable domain H-CDR 3 comprising SEQ IDNO:142.
 2. The antibody, or antigen-binding fragment thereof, accordingto claim 1, wherein said antibody or antigen-binding fragment thereofcomprises a heavy chain variable domain comprising SEQ ID NO:144.
 3. Theantibody, or antigen-binding fragment thereof, according to claim 1,wherein said antibody or antigen-binding fragment thereof comprises alight chain variable domain comprising SEQ ID NO:143.
 4. The antibody,or antigen-binding fragment thereof, according to claim 1, wherein theantibody or antigen-binding fragment thereof comprises both the heavychain variable domain comprising SEQ ID NO:144 and light chain variabledomain comprising SEQ ID NO:143.
 5. The antibody, or antigen-bindingfragment thereof, of claim 1, wherein the antigen-binding fragment isselected from the group consisting of an Fv fragment and a Fab-likefragment.
 6. The antibody, or antigen-binding fragment thereof, of claim1, wherein the antibody is selected from the group consisting of anantibody of subtypes IgG1, IgG2, IgG3 or IgG4.
 7. The antibody, orantigen-binding fragment thereof, of claim 1, wherein said antibody orantigen-binding fragment thereof binds to the D Region of Sortilin asdefined by SEQ ID NO:170.
 8. The antibody, or antigen-binding fragmentthereof, of claim 1, wherein said antibody or antigen-binding fragmentthereof binds to at least 3-7 consecutive amino acids within the DRegion of Sortilin as defined in SEQ ID NO:170.
 9. The antibody, orantigen-binding fragment thereof, of claim 1, wherein the antibody orantigen-binding fragment exhibits one or more of the followingproperties: (a) a binding affinity (KD) for Sortilin between 0.5-10 nM;(b) capability to reduce and/or inhibit PGRN binding to Sortilin; (c)capability to reduce and/or inhibit clearance of PGRN bySortilin-expressing cells; (d) capability to reduce and/or inhibit theendocytosis of PGRN by Sortilin-expressing cells; (e) capability toincrease the amount and/or concentration of PGRN in the brain, and/or(f) capability to increase the amount and/or concentration of PGRN inthe plasma in human-Sortilin-expressing knock-in mice.
 10. The antibody,or antigen-binding fragment thereof, of claim 1, wherein the antibody orantigen-binding fragment thereof is a human, humanized, recombinant orchimeric antibody.
 11. A pharmaceutical composition comprising anantibody, or an antigen-binding fragment thereof, of claim 1 and apharmaceutically-acceptable carrier.
 12. A method of treating a diseaseassociated with decreased PGRN levels in the brain of a patient,comprising administering an effective dosage of an antibody, or anantigen-binding fragment thereof, of claim
 1. 13. The method accordingto claim 12, wherein the disease is frontotemporal dementia, amyotrophiclateral sclerosis or Alzheimer's disease.
 14. A kit comprising anantibody, or antigen-binding fragment thereof, of claim
 1. 15. Anantibody, or antigen-binding fragment thereof, of claim 1 which has beenproduced or manufactured in a cell line comprising a mammal non-humancell line, an insect, yeast or bacterial cell line.
 16. The antibody, orantigen binding fragment thereof, of claim 15 produced in a CHO cellline, HEK cell line, BHK-21 cell line, murine cell line, myeloma cellline, fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAPcell line and HuH-7 human cell line.
 17. A method of making apharmaceutical composition comprising combining an antibody, or anantigen-binding fragment thereof, of claim 1, with apharmaceutically-acceptable carrier.
 18. A nucleic acid sequenceencoding an antibody or antibody binding fragment of claim
 1. 19. A cellline comprising the nucleic acid sequence of claim 18.